U.S. patent application number 10/228591 was filed with the patent office on 2003-10-02 for polypeptide with appetite regulating activity.
Invention is credited to Christiansen, Kennet, Hastrup, Sven, Judge, Martin Edward, Kristensen, Peter, Thim, Lars.
Application Number | 20030187187 10/228591 |
Document ID | / |
Family ID | 27512770 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030187187 |
Kind Code |
A1 |
Hastrup, Sven ; et
al. |
October 2, 2003 |
Polypeptide with appetite regulating activity
Abstract
The present invention relates to a polypeptide with appetite
regulating function/activity, a nucleic acid construct encoding the
polypeptide and a method of producing the polypeptide. The
invention further relates to recombinant vectors comprising the
nucleic acid construct encoding the polypeptide, recombinant host
cells comprising the nucleic acid construct or the recombinant
vector.
Inventors: |
Hastrup, Sven; (Copenhagen
N, DK) ; Christiansen, Kennet; (Rodovre, DK) ;
Thim, Lars; (Gentofte, DK) ; Judge, Martin
Edward; (Copenhagen K, DK) ; Kristensen, Peter;
(Bronshoj, DK) |
Correspondence
Address: |
Reza Green, Esq.
Novo Nordisk of North America, Inc.
Suite 6400
405 Lexington Avenue
New York
NY
10174-6401
US
|
Family ID: |
27512770 |
Appl. No.: |
10/228591 |
Filed: |
August 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10228591 |
Aug 26, 2002 |
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09048502 |
Mar 26, 1998 |
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6458927 |
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60044188 |
Apr 24, 1997 |
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60066527 |
Nov 25, 1997 |
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Current U.S.
Class: |
530/324 ;
435/320.1; 435/325; 435/326; 435/69.1; 530/388.1 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; C07K 14/475 20130101 |
Class at
Publication: |
530/324 ; 514/12;
435/69.1; 435/325; 435/320.1; 530/388.1; 435/326 |
International
Class: |
A61K 038/17; C12P
021/02; C12N 005/06; C07K 014/47; C07K 016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 1997 |
DK |
0358/97 |
Nov 19, 1997 |
DK |
1315/97 |
Claims
1. An isolated polypeptide comprising the sequence SEQ ID No.
1:
25 Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-
Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-
Glu-Leu-Pro-Arg-Arg-Gln-Leu-Arg-Ala-Pro-Gly-Ala-
Val-Leu-Gln-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-
Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-
Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-
Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-
Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-
Phe-Leu-Leu-Lys-Cys-Leu
in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the N-terminal end.
2. An isolated polypeptide comprising the sequence SEQ ID No.
2:
26 Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-
Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-
Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-
Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-
Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-
Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-IIe-Gly-Lys-Leu-
Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-
Leu-Leu-Lys-Cys-Leu
in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the n-terminal end:
3. An isolated polypeptide comprising the sequence SEQ ID No.
3:
27 Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-
Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-
Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-
Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Lys-Lys-
Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Cly-Glu-Gln-
Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-
Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-
Leu-Leu-Lys-Cys-Leu
in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the N-terminal end.
4. An isolated polypeptide comprising the sequence SEQ ID No.
4:
28 Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-
Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-
Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-
Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the N-terminal end.
5. An isolated polypeptide comprising the sequence SEQ ID No.
5:
29 Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-
Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-
Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-
Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
6. An isolated polypeptide comprising the sequence SEQ ID No.
6:
30 Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-
Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-
Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-
Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys- Leu
7. An isolated polypeptide comprising the sequence SEQ ID No.
7:
31 Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-
Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-
Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-
Phe-Leu-Leu-Lys-Cys-Leu
8. An isolated polypeptide comprising the sequence SEQ ID No.
8:
32 Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-
Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-
Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-
Leu-Leu-Lys-Cys-Leu
9. An isolated polypeptide comprising the sequence SEQ ID No.
9:
33 Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-
Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-
Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
10. An isolated polypeptide according to any one of the claims 5 to
9 in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the N-terminal end.
11. A nucleic acid construct comprising a nucleotide sequence
encoding a CART polypeptide or a fragment or variant thereof with
appetite regulating activity/function.
12. A nucleic acid construct according to claim 11 comprising a
nucleotide sequence encoding a polypeptide with a sequence selected
from the sequences SEQ ID Nos. 1 to 9.
13. A nucleic acid construct according to claim 12 comprising a
nucleotide sequence encoding a polypeptide with a sequence selected
from the sequences SEQ ID Nos. 1 to 9 in which the cysteine
residues are linked by disulphide bonds in the configuration I-III,
II-V and IV-VI when the cysteines are numbered from the N-terminal
end.
14. A recombinant vector comprising the nucleic acid construct
according to any one of the claims 11 to 13.
15. A recombinant host cell comprising the nucleic acid construct
according to any one of the claims 11 to 13 or the vector according
to claim 14.
16. The cell according to claim 15, which is of mammalian, insect,
plant, microbial, bacterial, or fungal origin.
17. A transgenic animal containing and expressing the nucleic acid
construct according to any one of the claims 11 to 13.
18. A transgenic plant containing and expressing the nucleic acid
construct according to any one of the claims 11 to 13.
19. A method of producing a CART polypeptide or a fragment or
variant thereof with appetite regulating activity/function, which
method comprises cultivating a cell according to claim 15 or 16 in
a suitable culture medium under conditions permitting expression of
the nucleic acid construct and recovering the resulting polypeptide
from the culture medium/cell.
20. A method of producing a CART polypeptide or a fragment or
variant thereof with appetite regulating activity/function, which
method comprises recovering the polypeptide produced by the
transgenic animal according to claim 17.
21. A method of producing a CART polypeptide or a fragment or
variant thereof with appetite regulating activity/function, which
method comprises growing a cell of a transgenic plant according to
claim 18, and recovering the polypeptide from the resulting
plant.
22. An antibody capable of specifically binding to a CART
polypeptide or a fragment or variant thereof with appetite
regulating activity/function.
23. An antibody capable of specifically binding to a polypeptide
with a sequence selected from the sequences SEQ ID Nos. 1 to 9.
24. An antibody capable of specifically binding to a polypeptide
with a sequence selected from the sequences SEQ ID Nos. 1 to 9 in
which the cysteine residues are linked by disulphide bonds in the
configuration I-III, II-V and IV-VI when the cysteines are numbered
from the N-terminal end.
25. An antibody according to any one of the claims 22 to 24 which
is a monoclonal antibody.
26. A hybridoma which produces a monoclonal antibody according to
claim 25.
27. An appetite regulating composition comprising a CART
polypeptide or a fragment or variant thereof and a pharmaceutical
acceptable carrier.
28. An appetite regulating composition according to claim 27
comprising a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 and a pharmaceutical acceptable
carrier.
29. An appetite regulating composition according to claim 28
comprising a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are
linked by disulphide bonds in the configuration I-III, II-V and
IV-VI when the cysteines are numbered from the N-terminal end and a
pharmaceutical acceptable carrier.
30. Use of a CART polypeptide or a fragment or variant thereof for
the preparation of a medicament for the regulation of appetite.
31. Use of a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 for the preparation of a medicament
for the regulation of appetite.
32. Use of a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are
linked by disulphide bonds in the configuration I-III, II-V and
IV-VI when the cysteines are numbered from the N-terminal end for
the preparation of a medicament for the regulation of appetite.
33. Use of a CART polypeptide or a fragment or variant thereof for
the preparation of a medicament for the treatment of obesity.
34. Use of a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 for the preparation of a medicament
for the treatment of obesity.
35. Use of a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are
linked by disulphide bonds in the configuration I-III, II-V and
IV-VI when the cysteines are numbered from the N-terminal end for
the preparation of a medicament for the treatment of obesity.
36. A method for the regulation of appetite comprising
administering to an subject in need thereof an effective amount of
an isolated CART polypeptide or a fragment or variant thereof.
37. A method for the regulation of appetite comprising
administering to an subject in need thereof an effective amount of
a polypeptide with a sequence selected from the sequences SEQ ID
Nos. 1 to 9.
38. A method for the regulation of appetite comprising
administering to an subject in need thereof an effective amount of
a polypeptide with a sequence selected from the sequences SEQ ID
Nos. 1 to 9 in which the cysteine residues are linked by disulphide
bonds in the configuration I-III, II-V and IV-VI when the cysteines
are numbered from the N-terminal end.
39. Any novel feature or combination of features described herein.
Description
FIELD OF INVENTION
[0001] The present invention relates to a polypeptide with appetite
regulating function/activity, a nucleic acid construct encoding the
polypeptide and a method of producing the polypeptide.
[0002] The invention further relates to recombinant vectors
comprising the nucleic acid construct encoding the polypeptide,
recombinant host cells comprising the nucleic acid construct or the
recombinant vector, a transgenic animal or plant containing and
expressing the nucleic acid construct, an appetite regulating
composition comprising the polypeptide, and the use of the
polypeptide for regulating appetite.
[0003] The polypeptide has appetite regulating activity/function in
mammals, including humans.
BACKGROUND OF THE INVENTION
[0004] It has been known that certain tumors when implanted into
rats after a period of growth suddenly induce severe anorexia and
adipsia (lack of eating and drinking) in the animal, whereas
closely related tumor lines do not, Madsen et al., Scand. J. Clin.
Invest. Supplement 220: 27-36.
[0005] The aim of this invention has been to find the factor(s)
responsible for this characteristic phenotype.
[0006] Cocaine and Amphetamine Regulated Transcript (CART) was
detected as one of several compounds that was selectively expressed
in anorectic versus non-anorectic secondary cultures of
glucagonomas. In situ hybridisation analysis of CART mRNA
expression has shown a decreased level of CART mRNA in the nucleus
arcuatus and nucleus paraventricularis of the rat hypothalamus
following fasting. Similarly, CART mRNA in the arcuate nucleus of
Zucker rats (fa/fa) was strongly decreased when compared to
heterozygote controls (fa/+) as measured by in situ hybridisation.
Thus, CART mRNA in the arcuate nucleus demonstrates a pattern of
change inverse to that known for NPY. The latter finding provides a
strong linkage between the expression of CART and biological
factors involved in food intake.
[0007] A polypeptide of at least 30 amino acids was found by Spiess
et al., 1981, Biochemistry 20:1982-1988 as an HPLC peak when
purifying somatostatine from sheep hypothalamus. The isolated
polypeptide was the C-terminal (IPI-CART) portion of CART. However,
no biological function was associated with this molecule.
[0008] The mature CART peptide has so far not been isolated and
characterised. A transcript to be upregulated in rat brain after
treatment with cocaine and amphetamine relating to CART was cloned.
This cloning indicates that the peptide may exist in a long form
consisting of 102 amino acid residues or in a short form consisting
of 89 amino acid residues (Douglass, J. et al. J. Neurosci. 15,
2471-2481, 1995).
[0009] The same group found the human gene and cDNA for CART. Only
the short form exists in humans (Douglass and Daoud (1996), Gene
169: 241-245).
[0010] In 1995 Amgen disclosed methods of reducing or preventing
neuron degeneration and promoting regeneration and restoration of
function induced by CART (WO 96/34619).
SUMMARY OF THE INVENTION
[0011] The aim of this invention has been to find the factor(s)
responsible for the above described characteristic phenotype.
[0012] It has now been found that a polypeptide with the sequence
SEQ ID No. 1 and fragments thereof have appetite regulating
activity/function:
1
Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-V-
al-Asp- Asp-Ala-Ser-His -Glu-Lys-Glu-Leu-Pro-Arg-Arg-Gln--
Leu-Arg-Ala-Pro-Gly-Ala- Val-Leu-Gln-Ile-Glu-Ala-Leu-Gln-G-
lu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-
Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Als-Gly-Glu-
Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-A-
sp-Cys-Pro-Arg Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-
-Leu
[0013] Furthermore, it has been found that the following
polypeptides have appetite regulating activity/function:
2 SEQ ID No. 2: Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-
-Asp-Ile-Tyr-Ser-Ala-Val-Asp- Asp-Ala-Ser-His-Glu-Lys-Glu-
-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-
Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-
Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-I-
le-Gly-Lys-Leu- Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Se-
r-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 3:
Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-
Asp-Als-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-V-
al-Leu-Lys-Lys- Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Ly-
s-Lys-Tyr-Gly-Gln-Val-Pro-Met- Cys-Asp-Ala-Gly-Glu-Gln-Cys-
-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-
Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
SEQ ID No. 4: Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-V-
al-Pro-Met-Cys-Asp-Ala-Gly-Glu- Gln-Cys-Ala-Val-Arg-Lys-G-
ly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-
Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 5:
Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-As-
p-Ala-Gly-Glu- Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gl-
y-Lys-Leu-Cys-Asp-Cys-Pro-Arg- Gly-Thr-Ser-Cys-Asn-Ser-Phe-
-Leu-Leu-Lys-Cys-Leu SEQ ID No. 6:
Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-
Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-C-
ys-Asp-Cys-Pro- Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Ly-
s-Cys-Leu SEQ ID No. 7: Lys-Tyr-Gly-Gln-Val-Pro-Me-
t-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-
Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-
Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 8:
Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Als-Gly-Glu-Cys-Ala-Val-Arg-Lys-Gly-
Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-C-
ys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu SEQ ID No. 9:
Cys-Asp-Als-Gly-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gl-
y-Lys-Leu- Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Ph-
e-Leu-Leu-Lys-Cys-Leu SEQ ID No.10:
Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-
Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-I-
le-Pro-Ile-Tyr- Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-As-
p-Ala-Gly-Glu-Gln-Cys-Ala-Val- Arg-Lys-Gly-Ala-Arg-Ile-Gly-
-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-
Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No.11:
Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-
Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-V-
al-Pro-Ile-Tyr- Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-As-
p-Ala-Gly-Glu-Gln-Cys-Ala-Val- Arg-Lys-Gly-Ala-Arg-Ile-Gly-
-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-
Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
[0014] The peptides SEQ ID Nos. 5 to 11 are considered to be novel
per se and are constituting a part of the invention.
[0015] In a preferred embodiment of the present invention the
cysteine residues of the above peptides SEQ ID Nos. 1 to 11 are
linked by disulphide bonds in the configuration I-III, II-V and
IV-VI when the cysteines are numbered from the N-terminal. These
peptides are also considered to be novel per se and are
constituting a part of the invention.
[0016] In the present context, the term "appetite regulating
activity/function" is intended to mean any activity/function which
suppresses appetite e.g. by inducing a feeling of satiety or by
inhibiting the sensation of hunger. The appetite regulating
activity/function may be measured according to the test methods
described in Example 9 or 20
[0017] In another aspect, the invention relates to nucleic acid
constructs comprising a nucleotide sequence encoding a CART
polypeptide or a fragment or variant thereof with appetite
regulating activity/function.
[0018] In a further aspect, the invention relates to nucleic acid
constructs encoding a polypeptide with a sequence selected from the
sequences SEQ ID Nos. 1 to 9 such as the sequences SEQ ID Nos. 1 to
9 in which the cysteine residues are linked by disulphide bonds in
the configuration I-III, II-V and IV-VI when the cysteines are
numbered from the N-terminal end.
[0019] In a further aspect, the invention relates to recombinant
vectors comprising the nucleic acid constructs and recombinant host
cells comprising the nucleic acid constructs or the vectors.
[0020] In a further aspect, the invention relates to a method of
producing a CART polypeptide or a fragment or a variant thereof
with appetite regulating activity/function which method comprises
cultivating a host cell as defined above in a suitable culture
medium under conditions permitting expression of the nucleic acid
construct and recovering the resulting polypeptide from the culture
medium/cell.
[0021] In a further aspect, the invention relates to transgenic
animals or transgenic plants comprising the nucleic acid construct
as defined above as well as methods of producing a CART polypeptide
or a fragment or a variant thereof with appetite regulating
activity/function using such transgenic animals or transgenic
plants.
[0022] In still a further aspect, the invention relates to an
antibody capable of specifically binding to a CART polypeptide or a
fragment or a variant thereof with appetite regulating
activity/function, such as a polypeptide with a sequence selected
from the sequences SEQ ID Nos. 1 to 9, e.g. the sequences SEQ ID
Nos. 1 to 9 in which the cysteine residues are linked by disulphide
bonds in the configuration I-III, II-V and IV-VI when the cysteines
are numbered from the N-terminal end. In a preferred embodiment of
the invention the antibody is monoclonal and the invention
furthermore relates to hybridomas producing such monoclonal
antibodies.
[0023] In a further aspect, the invention relates to appetite
regulating compositions comprising the polypeptides as defined
above and a pharmaceutically acceptable carrier and the use of the
polypeptides for the preparation of medicaments for the regulation
of appetite. In a preferred embodiment of the invention the
medicaments are used for the treatment of obesity.
[0024] Furthermore, the invention relates to a method for the
regulation of appetite comprising administering to a subject in
need thereof an effective amount of a polypeptide as defined above.
In a further aspect, the invention relates to the use of CART or
CART fragments or variants to identify a functional receptor and
the subsequent use of the CART receptor to identify receptor
agonists with appetite regulating activity.
[0025] In a further aspect, the invention relates to compounds that
upregulate the CART expression and thereby regulate appetite.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1: Cloning of CART.
[0027] FIG. 2. Expression in E. coli. Fusion to
Glutathione-S-transferase.
[0028] FIG. 3: Expression in E. coli. Fusion to Thioredoxin.
[0029] FIG. 4: Expression of CART in yeast.
[0030] FIG. 5: Sequence of heterologous protein expression cassette
of plasmid pEA182 and pEA183 in yeast.
[0031] FIG. 6: Preparation of FXa-digest of the thioredoxin-CART
fusion protein.
[0032] FIG. 7: Analytical HPLC of CART yeast supernatant H-372.
[0033] FIG. 8: SP-Sepharose Column.
[0034] FIG. 9: Analytical HPLC of CART fragment, pool A.
[0035] FIG. 10: Analytical HPLC of CART fragment, pool B.
[0036] FIG. 11: Analytical HPLC of CART fragment, pool C.
[0037] FIG. 12: Primary and secondary structure of "IPI-CART"
showing the I-III, II-V and IV-VI disulphide bond
configuration.
[0038] FIG. 13: Effect of fasting on CART mRNA expression.
[0039] FIG. 14: CART mRNA in Zucker rat arcuate nucleus and
heterozygote controls.
[0040] FIG. 15: S. cerevisiae plasmid for the expression and
secretion of Glu-Glu-Ile-Asp-CART(55-102). TPI-prom. and TPI-term.
are S. cerevisiae triosephosphate isomerase transcription promoter
and terminator sequences, respectively. TPI S. pombe is the
Schizosaccharmyces pombe triosephosphate isomerase gene. Only
restriction sites relevant for the plasmid construction have been
indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0041] This invention is based on the unexpected and surprising
discovery that CART polypeptide has been found to possess appetite
regulating function/activity. In the present context the term
"polypeptide" is understood to include a mature protein or a
precursor form thereof as well as a functional fragment thereof
which essentially has the activity of the full-length
polypeptide.
[0042] Furthermore, the term "polypeptide" is intended to include
homologues of said polypeptide. Such homologues comprise an amino
acid sequence exhibiting a degree of identity of at least 60%,
preferably 80% with the amino acid sequences shown in SEQ ID Nos.
1-9. The degree of identity may be determined by conventional
methods, see for instance, Altshul et al., Bull. Math. Bio. 48:
603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are
aligned to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the "blosum 62"
scoring matrix of Henikoff and Henikoff, supra.
[0043] Alternatively, the homologue of the polypeptide may be one
encoded by a nucleotide sequence hybridizing with an
oligonucleotide probe prepared on the basis of the polypeptide
sequences shown in SEQ ID Nos. 1-9 .
[0044] In a further aspect the invention relates to a variant of
the polypeptide of the invention. The variant is one in which one
or more amino acid residues in one or more positions have been
substituted by other amino acid residues.
[0045] Homologues of the present polypeptide may have one or more
amino acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein, small deletions, typically of one to about
30 amino acids, small amino- or carboyxyl-terminal extensions, such
as an amino-terminal methionine residue, a small linker peptide of
up to about 20-25 residues, or a small extension that facilitates
purification, such as a poly-histidine tract, an antigenic epitope
or a binding domain. See in general Ford et al., Protein Expression
and Purification 2: 95-107, 1991. Examples of conservative
substitutions are within the group of basic amino acids (such as
arginine, lysine, histidine), acidic amino acids (such as glutamic
acid and aspartic acid), polar amino acids (such as glutamine and
asparagine), hydrophobic amino acids (such as leucine, isoleucine,
valine), aromatic amino acids (such as phenylalanine, tryptophan,
tyrosine) and small amino acids (such as glycine, alanine, serine,
threonine, methionine).
[0046] It will be apparent to persons skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acids essential to the activity of the polypeptide of the
invention, and therefore preferably not subject to substitution,
may be identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, Science 244, 1081-1085, 1989). In the latter
technique mutations are introduced at every residue in the
molecule, and the resultant mutant molecules are tested for
biological activity (e.g. appetite regulation) to identify amino
acid residues that are critical to the activity of the molecule.
Sites of ligand-receptor interaction can also be determined by
analysis of crystal structure as determined by such techniques as
nuclear magnetic resonance, crystallography or photoaffinity
labelling. See, for example, de Vos et al., Science 255: 306-312,
1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et
al., FEBS Lett. 309: 59-64, 1992.
[0047] The homologue may be an allelic variant, i.e. an alternative
form of a gene that arises through mutation, or an altered
polypeptide encoded by the mutated gene, but having substantially
the same activity as the polypeptide of the invention. Hence
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequence.
[0048] The homologue of the present polypeptide may also be a
species homologue, i.e. a polypeptide with a similar activity
derived from another mammalian species eg. rat, mouse, sheep or
human.
[0049] Furthermore, homologues of said polypeptide may be found in
other tisssues such as the brain and pancreas.
[0050] A homologue of the polypeptide may be isolated by preparing
a genomic or cDNA library of a cell of the species or tissue in
question, and screening for DNA sequences coding for all or part of
the homologue by using synthetic oligonucleotide probes in
accordance with standard techniques, e.g. as described by Sambrook
et al., Molecular Cloning:A Laboratory Manual, 2nd. Ed. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, or by means of
polymerase chain reaction (PCR) using specific primers as described
by Sambrook et al. and Saiki at al., Science 239 (1988)
487-491.
[0051] It may be preferred to provide the polypeptide in a highly
purified form, i.e. greater than 90% pure, more preferably 95% and
most preferably 99% pure, as determined by analytical HPLC.
[0052] The currently preferred polypeptides of the invention are
the ones comprising the amino acid sequences shown in SEQ ID Nos.
1-9.
[0053] Nucleic Acid Construct
[0054] As used herein the term "nucleic acid construct" is intended
to indicate any nucleic acid molecule of cDNA, genomic DNA,
synthetic DNA or RNA origin. The term "construct" is intended to
indicate a nucleic acid segment which may be single or double
stranded, and which may be based on a complete or partial naturally
occurring nucleotide sequence encoding a polypeptide of interest.
The construct may optionally contain other nucleic acid
segments.
[0055] The nucleic acid construct of the invention encoding the
polypeptide of the invention may suitably be of genomic or cDNA
origin, for instance obtained by preparing a genomic or cDNA
library and screening for DNA sequences coding for all or part of
the polypeptide by hybridization using synthetic oligonucleotide
probes in accordance with standard techniques (cf. Sambrook et al.,
supra). For the present purpose, the DNA sequence encoding the
polypeptide is preferably of mammalian origin, i.e. derived from a
genomic DNA or cDNA library. More preferably, the DNA sequence may
be of rodent origin, e.g. rat or mice origin. Even more preferably,
the DNA sequence may be of human origin.
[0056] The nucleic acid construct of the invention encoding the
polypeptide may also be prepared synthetically by established
standard methods, e.g. the phosphoamidite method described by
Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869,
or the method described by Matthes et al., EMBO Journal 3 (1984),
801-805. According to the phosphoamidite method, oligonucleotides
are synthesized, e.g. in an automatic DNA synthesizer, purified,
annealed, ligated and cloned in suitable vectors.
[0057] Furthermore, the nucleic acid construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or mixed genomic
and cDNA origin prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate), the fragments
corresponding to various parts of the entire nucleic acid
construct, in accordance with standard techniques.
[0058] The nucleic acid construct may also be prepared by
polymerase chain reaction using specific primers, for instance as
described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239
(1988), 487-491.
[0059] As template for the PCR cloning we used the same double
stranded cDNA preparation as described in example 1 (from
MSL-A-AN).
3 The PCR reaction, 25 cycles: 60 sec 94.degree. C. 30 sec
52.degree. C. 60 sec 72.degree. C.
[0060] The nucleic acid construct is preferably a DNA construct
which term will be used exclusively in the following.
Recombinant Vector
[0061] In a further aspect, the present invention relates to a
recombinant vector comprising a DNA construct of the invention. The
recombinant vector into which the DNA construct of the invention is
inserted may be any vector which may conveniently be subjected to
recombinant DNA procedures, and the choice of vector will often
depend on the host cell into which it is to be introduced. Thus,
the vector may be an autonomously replicating vector, i.e. a vector
which exists as an extrachromosomal entity, the replication of
which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a
host cell, is integrated into the host cell genome and replicated
together with the chromosome(s) into which it has been
integrated.
[0062] The vector is preferably an expression vector in which the
DNA sequence encoding the polypeptide of the invention is operably
linked to additional segments required for transcription of the
DNA. In general, the expression vector is derived from plasmid or
viral DNA, or may contain elements of both. The term, "operably
linked" indicates that the segments are arranged so that they
function in concert for their intended purposes, e.g. transcription
initiates in a promoter and proceeds through the DNA sequence
coding for the polypeptide.
[0063] The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell.
[0064] Examples of suitable promoters for directing the
transcription of the DNA encoding the polypeptide of the invention
in mammalian cells are the SV40 promoter (Subramani et al., Mol.
Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene)
promoter (Palmiter et al., Science 222 (1983), 809-814) or the
adenovirus 2 major late promoter.
[0065] An example of a suitable promoter for use in insect cells is
the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al.,
FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al.,
J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica
polyhedrosis virus basic protein promoter (EP 397 485), the
baculovirus immediate early gene 1 promoter (U.S. Pat. No.
5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K
delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.
5,162,222).
[0066] Examples of suitable promoters for use in yeast host cells
include promoters from yeast glycolytic genes (Hitzeman et al., J.
Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol.
Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young
et al., in Genetic Engineering of Microorganisms for Chemicals
(Hollaender et al, eds.), Plenum Press, N.Y., 1982), or the TPI1
(U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature 304
(1983), 652-654) promoters.
[0067] Examples of suitable promoters for use in filamentous fungus
host cells are, for instance, the ADH3 promoter (McKnight et al.,
The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of
other useful promoters are those derived from the gene encoding A.
oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.
niger neutral .alpha.-amylase, A. niger acid stable
.alpha.-amylase, A. niger or A. awamori glucoamylase (gluA),
Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae
triose phosphate isomerase or A. nidulans acetamidase. Preferred
are the TAKA-amylase and gluA promoters.
[0068] Examples of suitable promoters for use in bacterial host
cells include the promoter of the Bacillus stearothermophilus
maltogenic amylase gene, the Bacillus licheniformis alpha-amylase
gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus
subtilis alkaline protease gen, or the Bacillus subtilis xylosidase
gene, or by the phage Lambda P.sub.R or P.sub.L promoters or the E.
coli lac, trp or tac promoters.
[0069] The DNA sequence encoding the polypeptide of the invention
may also, if necessary, be operably connected to a suitable
terminator, such as the human growth hormone terminator (Palmiter
et al. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki,
op. cit.) or ADH3 (McKnight et al., op. cit.) terminators. The
vector may further comprise elements such as polyadenylation
signals (e.g. from SV40 or the adenovirus 5 Elb region),
transcriptional enhancer sequences (e.g. the SV40 enhancer) and
translational enhancer sequences (e.g. the ones encoding adenovirus
VA RNAs).
[0070] The recombinant vector of the invention may further comprise
a DNA sequence enabling the vector to replicate in the host cell in
question. An example of such a sequence (when the host cell is a
mammalian cell) is the SV40 origin of replication.
[0071] When the host cell is a yeast cell, suitable sequences
enabling the vector to replicate are the yeast plasmid 2.mu.
replication genes REP 1-3 and origin of replication.
[0072] When the host cell is a bacterial cell, sequences enabling
the vector to replicate are e.g. the Col E1 origin of replication
as in pUC19 or pBR322 or the p15A origin of replication as in
pACYC184 when the bacterium is E. coli. When the bacterium is B.
subtilis the origin of replication from e.g. pUB110 is often
used.
[0073] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
such as the gene coding for dihydrofolate reductase (DHFR) or the
Schizosaccharomyces pombe TPI gene (described by P. R. Russell,
Gene 40, 1985, pp. 125-130), or one which confers resistance to a
drug, e.g. ampicillin, kanamycin, tetracycline, chloramphenicol,
neomycin, hygromycin or methotrexate. For filamentous fungi,
selectable markers include amdS, pyrG, argB, niaD, sC.
[0074] To direct a polypeptide of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
may be provided in the recombinant vector. The secretory signal
sequence is joined to the DNA sequence encoding the polypeptide in
the correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the polypeptide. The
secretory signal sequence may be that normally associated with the
polypeptide or may be from a gene encoding another secreted
protein.
[0075] For secretion from yeast cells, the secretory signal
sequence may encode any signal peptide which ensures efficient
direction of the expressed polypeptide into the secretory pathway
of the cell. The signal peptide may be naturally occurring signal
peptide, or a functional part thereof, or it may be a synthetic
peptide. Suitable signal peptides have been found to be the
.alpha.-factor signal peptide (cf. U.S. Pat. No. 4,870,008), the
signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et
al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase
signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp.
887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the
yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani
et al., Yeast 6, 1990, pp. 127-137).
[0076] For efficient secretion in yeast, a sequence encoding a
leader peptide may also be inserted downstream of the signal
sequence and upstream of the DNA sequence encoding the polypeptide.
The function of the leader peptide is to allow the expressed
polypeptide to be directed from the endoplasmic reticulum to the
Golgi apparatus and further to a secretory vesicle for secretion
into the culture medium (i.e. exportation of the polypeptide across
the cell wall or at least through the cellular membrane into the
periplasmic space of the yeast cell). The leader peptide may be the
yeast .alpha.-factor leader (the use of which is described in e.g.
U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP
163 529). Alternatively, the leader peptide may be a synthetic
leader peptide, which is to say a leader peptide not found in
nature. Synthetic leader peptides may, for instance, be constructed
as described in WO 89/02463 or WO 92/11378.
[0077] For use in filamentous fungi, the signal peptide may
conveniently be derived from a gene encoding an Aspergillus sp.
amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase
or protease, or a gene encoding a Humicola lanuginosa lipase. The
signal peptide is preferably derived from a gene encoding A. oryzae
TAKA amylase, A. niger neutral .alpha.-amylase, A. niger
acid-stable amylase, or A. niger glucoamylase.
[0078] For use in insect cells, the signal peptide may conveniently
be derived from an insect gene (cf. WO 90/05783), such as the
lepidopteran Manduca sexta adipokinetic hormone precursor signal
peptide (cf. U.S. Pat. No. 5,023,328).
[0079] The procedures used to ligate the DNA sequences coding for
the present polypeptide, the promoter and optionally the terminator
and/or secretory signal sequence, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., op.cit.).
[0080] Host Cells
[0081] The DNA sequence encoding the present polypeptide introduced
into the host cell may be either homologous or heterologous to the
host in question. If homologous to the host cell, i.e. produced by
the host cell in nature, it will typically be operably connected to
another promoter sequence or, if applicable, another secretory
signal sequence and/or terminator sequence than in its natural
environment. The term "homologous" is intended to include a cDNA
sequence encoding a polypeptide native to the host organism in
question. The term "heterologous" is intended to include a DNA
sequence not expressed by the host cell in nature. Thus, the DNA
sequence may be from another organism, or it may be a synthetic
sequence.
[0082] The host cell into which the DNA construct or the
recombinant vector of the invention is introduced may be any cell
which is capable of producing the present polypeptide and includes
bacteria, yeast, fungi and higher eukaryotic cells.
[0083] Examples of bacterial host cells which, on cultivation, are
capable of producing the polypeptide of the invention are
grampositive bacteria such as strains of Bacillus, such as strains
of B. subtilis, B. licheniformis, B. lentus, B. brevis, B.
stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.
coagulans, B. circulans, B. lautus, B. megatherium or B.
thuringiensis, or strains of Streptomyces, such as S. lividans or
S. murinus, or gramnegative bacteria such as Echerichia coli. The
transformation of the bacteria may be effected by protoplast
transformation or by using competent cells in a manner known per se
(cf. Sambrook et al., supra).
[0084] When expressing the polypeptide in bacteria such as E. coli,
the polypeptide may be retained in the cytoplasm, typically as
insoluble granules (known as inclusion bodies), or may be directed
to the periplasmic space by a bacterial secretion sequence. In the
former case, the cells are lysed and the granules are recovered and
denatured after which the polypeptide is refolded by diluting the
denaturing agent. In the latter case, the polypeptide may be
recovered from the periplasmic space by disrupting the cells, e.g.
by sonication or osmotic shock, to release the contents of the
periplasmic space and recovering the polypeptide.
[0085] Examples of suitable mammalian cell lines are the COS (ATCC
CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or
CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian
cells and expressing DNA sequences introduced in the cells are
described in e.g. Kaufmnan and Sharp, J. Mol. Biol. 159 (1982),
601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341;
Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426;
Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic
Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52
(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.
[0086] Examples of suitable yeasts cells include cells of
Saccharomyces spp. or Schizosaccharomyces spp., in particular
strains of Saccharomyces cerevisiae or Saccharomyces kluyveri.
Methods for transforming yeast cells with heterologous DNA and
producing heterologous polypeptides therefrom are described, e.g.
in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. No.
4,870,008, U.S. Pat. No. 5,037,743, and U.S. Pat. No. 4,845,075,
all of which are hereby incorporated by reference. Transformed
cells are selected by a phenotype determined by a selectable
marker, commonly drug resistance or the ability to grow in the
absence of a particular nutrient, e.g. leucine. A preferred vector
for use in yeast is the POT1 vector disclosed in U.S. Pat. No.
4,931,373. The DNA sequence encoding the polypeptide of the
invention may be preceded by a signal sequence and optionally a
leader sequence , e.g. as described above. Further examples of
suitable yeast cells are strains of Kluyveromyces, such as K.
lactis, Hansenula, e.g. H. polymorpha, or Pichia, e.g. P. pastoris
(cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465;
U.S. Pat. No. 4,882,279).
[0087] Examples of other fungal cells are cells of filamentous
fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or
Trichoderma spp., in particular strains of A. oryzae, A. nidulans
or A. niger. The use of Aspergillus spp. for the expression of
proteins is described in, e.g., EP 272 277 and EP 230 023. The
transformation of F. oxysporum may, for instance, be carried out as
described by Malardier et al., 1989, Gene 78: 147-156.
[0088] When a filamentous fungus is used as the host cell, it may
be transformed with the DNA construct of the invention,
conveniently by integrating the DNA construct in the host
chromosome to obtain a recombinant host cell. This integration is
generally considered to be an advantage as the DNA sequence is more
likely to be stably maintained in the cell. Integration of the DNA
constructs into the host chromosome may be performed according to
conventional methods, e.g. by homologous or heterologous
recombination.
[0089] Transformation of insect cells and production of
heterologous polypeptides therein may be performed as described in
U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos.
5,155,037; 5,162,222; EP 397,485; all of which are incorporated
herein by reference. The insect cell line used as the host may
suitably be a Lepidoptera cell line, such as Spodoptera frugiperda
cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214).
Culture conditions may suitably be as described in, for instance,
WO 89/01029 or WO 89/01028, or any of the aforementioned
references.
[0090] The transformed or transfected host cell described above is
then cultured in a suitable nutrient medium under conditions
permitting the expression of the present polypeptide, after which
the resulting polypeptide is recovered from the culture.
[0091] The medium used for culturing the cells may be any
conventional medium suitable for growing the host cells, such as
minimal or complex media containing appropriate supplements.
Suitable media are available from commercial suppliers or may be
prepared according to published recipes (e.g. in catalogues of the
American Type Culture Collection). The polypeptide produced by the
cells may then be recovered from the culture medium by conventional
procedures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gelfiltration
chromatography, affinity chromatography, or the like, dependent on
the type of polypeptide in question.
[0092] Transgenic Animals
[0093] It is also within the scope of the present invention to
employ transgenic animal technology to produce the present
polypeptide. A transgenic animal is one in whose genome a
heterologous DNA sequence has been introduced. In particular, the
polypeptide of the invention may be expressed in the mammary glands
of a non-human female mammal, in particular one which is known to
produce large quantities of milk. Examples of preferred mammals are
livestock animals such as goats, sheep and cattle, although smaller
mammals such as mice, rabbits or rats may also be employed.
[0094] The DNA sequence encoding the present polypeptide may be
introduced into the animal by any one of the methods previously
described for the purpose. For instance, to obtain expression in a
mammary gland, a transcription promoter from a milk protein gene is
used. Milk protein genes include the genes encoding casein (cf.
U.S. Pat. No. 5,304,489), beta-lactoglobulin, alpha-lactalbumin and
whey acidic protein. The currently preferred promoter is the
beta-lactoglobulin promoter (cf. Whitelaw et al., Biochem J. 286,
1992, pp. 31-39). It is generally recognized in the art that DNA
sequences lacking introns are poorly expressed in transgenic
animals in comparison with those containing introns (cf. Brinster
et al., Proc. Natl. Acad. Sci. USA 85, 1988, pp. 836-840; Palmiter
et al., Proc. Natl. Acad. Sci. USA 88, 1991, pp. 478-482; Whitelaw
et al., Transgenic Res. 1, 1991, pp. 3-13; WO 89/01343; WO
91/02318). For expression in transgenic animals, it is therefore
preferred, whenever possible, to use genomic sequences containing
all or some of the native introns of the gene encoding the
polypeptide of interest. It may also be preferred to include at
least some introns from, e.g. the beta-lactoglobulin gene. One such
region is a DNA segment which provides for intron splicing and RNA
polyadenylation from the 3' non-coding region of the ovine
beta-lactogloblin gene. When substituted for the native 3'
non-coding sequences of a gene, this segment will enhance and
stabilise expression levels of the polypeptide of interest. It may
also be possible to replace the region surrounding the initiation
codon of the polypeptide of interest with corresponding sequences
of a milk protein gene. Such replacement provides a putative
tissue-specific initiation environment to enhance expression.
[0095] For expression of the present polypeptide in transgenic
animals, a nucleotide sequence encoding the polypeptide is operably
linked to additional DNA sequences required for its expression to
produce expression units. Such additional sequences include a
promoter as indicated above, as well as sequences providing for
termination of transcription and polyadenylation of mRNA. The
expression unit further includes a DNA sequence encoding a
secretory signal sequence operably linked to the sequence encoding
the polypeptide. The secretory signal sequence may be one native to
the polypeptide or may be that of another protein such as a milk
protein (cf. von Heijne et al., Nucl. Acids Res. 14, 1986, pp.
4683-4690; and U.S. Pat. No. 4,873,316).
[0096] Construction of the expression unit for use in transgenic
animals may conveniently be done by inserting a DNA sequence
encoding the present polypeptide into a vector containing the
additional DNA sequences, although the expression unit may be
constructed by essentially any sequence of ligations. It is
particularly convenient to provide a vector containing a DNA
sequence encoding a milk protein and to replace the coding region
for the milk protein with a DNA sequence coding for the present
polypeptide, thereby creating a fusion which includes expression
control sequences of the milk protein gene.
[0097] The expression unit is then introduced into fertilized ova
or early-stage embryos of the selected host species. Introduction
of heterologous DNA may be carried out in a number of ways,
including microinjection (cf. U.S. Pat. No. 4,873,191), retroviral
infection (cf. Jaenisch, Science 240, 1988, pp. 1468-1474) or
site-directed integration using embryonic stem cells (reviewed by
Bradley et al., Bio/Technology 10, 1992, pp. 534-539). The ova are
then implanted into the oviducts or uteri of pseudopregnant females
and allowed to develop to term. Offspring carrying the introduced
DNA in their germ line can pass the DNA on to their progeny,
allowing the development of transgenic herds.
[0098] General procedures for producing transgenic animals are
known in the art, cf. for instance, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory,
1986; Simons et al., Bio/Technology 6, 1988, pp. 179-183; Wall et
al., Biol. Reprod. 32, 1985, pp. 645-651; Buhler et al.,
Bio/Technology 8, 1990, pp. 140-143; Ebert et al., Bio/Technology
6: 179-183, 1988; Krimpenfort et al., Bio/Tecnology 9: 844-847,
1991, Wall et al., J. Cell. Biochem. 49: 113-120, 1992; U.S. Pat.
No. 4,873,191, U.S. Pat. No. 4,873,316; WO 88/00239, WO 90/05188;
WO 92/11757 and GB 87/00458. Techniques for introducing
heterologous DNA sequences into mammals and their germ cells were
originally developed in the mouse. See, e.g. Gordon et al., Proc.
Natl. Acad. Sci. USA 77: 7380-7384, 1980, Gordon and Ruddle,
Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41:
343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82:
4438-4442, 1985; and Hogan et al. (ibid.). These techniques were
subsequently adapted for use with larger animals, including
livestock species (see e.g., WO 88/00239, WO 90/01588 and WO
92/11757; and Simons et al., Bio/Technology 6: 179-183, 1988). To
summarize, in the most efficient route used to date in the
generation of transgenic mice or livestock, several hundred linear
molecules of the DNA of interest are injected into one of the
pro-nuclei of a fertilized egg according to techniques which have
become standard in the art. Injection of DNA into the cytoplasm of
a zygote can also be employed.
[0099] Transgenic Plants
[0100] Production in transgenic plants may also be employed.
[0101] It has previously been described to introduce DNA sequences
into plants, which sequences code for protein products imparting to
the transformed plants certain desirable properties such as
increased resistance against pests, pathogens, herbicides or stress
conditions (cf. for instance EP 90 033, EP 131 620, EP 205 518, EP
270 355, WO 89/04371 or WO 90/02804), or an improved nutrient value
of the plant proteins (cf. for instance EP 90 033, EP 205 518 or WO
89/04371). Furthermore, WO 89/12386 discloses the transformation of
plant cells with a gene coding for levansucrase or dextransucrase,
regeneration of the plant (especially a tomato plant) from the cell
resulting in fruit products with altered viscosity
characteristics.
[0102] In the plant cell, the DNA sequence encoding the present
polypeptide is under the control of a regulatory sequence which
directs the expression of the polypeptide from the DNA sequence in
plant cells and intact plants. The regulatory sequence may be
either endogenous or heterologous to the host plant cell.
[0103] The regulatory sequence may comprise a promoter capable of
directing the transcription of the DNA sequence encoding the
polypeptide in plants. Examples of promoters which may be used
according to the invention are the 35s RNA promoter from
cauliflower mosaic virus CaMV), the class I patatin gene B 33
promoter, the ST-LS1 gene promoter, promoters conferring
seed-specific expression, e.g. the phaseolin promoter, or promoters
which are activated on wounding, such as the promoter of the
proteinase inhibitor II gene or the wun1 or wun2 genes.
[0104] The promoter may be operably connected to an enhancer
sequence, the purpose of which is to ensure increased transcription
of the DNA sequence encoding the polypeptide. Examples of useful
enhancer sequences are enhancers from the 5'-upstream region of the
35s RNA of CaMV, the 5'-upstream region of the ST-LS1 gene, the
5'-upstream region of the Cab gene from wheat, the 5'-upstream
region of the 1'- and 2'-genes of the T.sub.R-DNA of the Ti plasmid
pTi ACH5, the 5'-upstream region of the octopine synthase gene, the
5'-upstream region of the leghemoglobin gene, etc.
[0105] The regulatory sequence may also comprise a terminator
capable of terminating the transcription of the DNA sequence
encoding the polypeptide in plants. Examples of suitable
terminators are the terminator of the octopine synthase gene of the
T-DNA of the Ti-plasmid pTiACH5 of Agrobacterium tumefaciens, of
the gene 7 of the T-DNA of the Ti plasmid pTiACH5, of the nopaline
synthase gene, of the 35s RNA-coding gene from CaMV or from various
plant genes, e.g. the ST-LS1 gene, the Cab gene from wheat, class I
and class II patatin genes, etc.
[0106] The DNA sequence encoding the polypeptide may also be
operably connected to a DNA sequence encoding a leader peptide
capable of directing the transport of the expressed polypeptide to
a specific cellular compartment (e.g. vacuoles) or to extracellular
space. Examples of suitable leader peptides are the leader peptide
of proteinase inhibitor II from potato, the leader peptide and an
additional about 100 amino acid fragments of patatin, or the
transit peptide of various nucleus-encoded proteins directed into
chloroplasts (e.g. from the St-LS1 gene, SS-Rubisco genes, etc.) or
into mitochondria (e.g. from the ADP/ATP translocator).
[0107] Furthermore, the DNA sequence encoding the polypeptide may
be modified in the 5' non-translated region resulting in enhanced
translation of the sequence. Such modifications may, for instance,
result in removal of hairpin loops in RNA of the 5' non-translated
region. Translation enhancement may be provided by suitably
modifying the omega sequence of tobacco mosaic virus or the leaders
of other plant viruses (e.g. BMV, MSV) or of plant genes expressed
at high levels (e.g. SS-Rubisco, class I patatin or proteinase
inhibitor II genes from potato).
[0108] The DNA sequence encoding the polypeptide may furthermore be
connected to a second DNA sequence encoding another polypeptide or
a fragment thereof in such a way that expression of said DNA
sequences results in the production of a fusion protein. When the
host cell is a potato plant cell, the second DNA sequence may, for
instance, encode patatin or a fragment thereof (such as a fragment
of about 100 amino acids).
[0109] The plant in which the DNA sequence coding for the
polypeptide is introduced may suitably be a dicotyledonous plant,
examples of which are is a tobacco, potato, tomato, or leguminous
(e.g. bean, pea, soy, alfalfa) plant. It is, however, contemplated
that mono-cotyledonous plants, e.g. cereals, may equally well be
transformed with the DNA sequence coding for the enzyme.
[0110] Procedures for the genetic manipulation of monocotyledonous
and dicotyledonous plants are well known. In order to construct
foreign genes for their subsequent introduction into higher plants,
numerous cloning vectors are available which generally contain a
replication system for E. coli and a selectable/screenable marker
system permitting the recognition of transformed cells. These
vectors include e.g. pBR322, the pUC series, pACYC, M13 mp series
etc. The foreign sequence may be cloned into appropriate
restriction sites. The recombinant plasmid obtained in this way may
subsequently be used for the transformation of E. coli. Transformed
E. coli cells may be grown in an appropriate medium, harvested and
lysed. The chimeric plasmid may then be reisolated and analyzed.
Analysis of the recombinant plasmid may be performed by e.g.
determination of the nucleotide sequence, restriction analysis,
electrophoresis and other molecular-biochemical methods. After each
manipulation the sequence may be cleaved and ligated to another DNA
sequence. Each DNA sequence can be cloned on a separate plasmid
DNA. Depending on the way used for transferring the foreign DNA
into plant cells other DNA sequences might be of importance. In
case the Ti-plasmid or the Ri plasmid of Agrobacterium tumefaciens
or Agrobacterium rhizogenes, at least the right border of the T-DNA
may be used, and often both the right and the left borders of the
T-DNA of the Ri or Ti plasmid will be present flanking the DNA
sequence to be transferred into plant cells.
[0111] The use of the T-DNA for transferring foreign DNA into plant
cells has been described extensively in the prior literature (cf.
Gasser and Fraley, 1989, Science 244, 1293-1299 and references
cited therein). After integration of the foreign DNA into the plant
genome, this sequence is fairly stable at the original locus and is
usually not lost in subsequent mitotic or meiotic divisions. As a
general rule, a selectable marker gene will be cotransferred in
addition to the gene to be transferred, which marker renders the
plant cell resistant to certain antibiotics, e.g. kanamycin,
hygromycin, G418 etc. This marker permits the recognition of the
transformed cells containing the DNA sequence to be transferred
compared to nontransformed cells.
[0112] Numerous techniques are available for the introduction of
DNA into a plant cell. Examples are the Agrobacterium mediated
transfer, the fusion of protoplasts with liposomes containing the
respective DNA, microinjection of foreign DNA, electroporation etc.
In case Agrobacterium mediated gene transfer is employed, the DNA
to be transferred has to be present in special plasmids which are
either of the intermediate type or the binary type. Due to the
presence of sequences homologous to T-DNA sequences, intermediate
vectors may integrate into the Ri- or Ti-plasmid by homologous
recombination. The Ri- or Ti-plasmid additionally contains the
vir-region which is necessary for the transfer of the foreign gene
into plant cells. Intermediate vectors cannot replicate in
Agrobacterium species and are transferred into Agrobacterium by
either direct transformation or mobilization by means of helper
plasmids (conjugation). (Cf. Gasser and Fraley, op. cit. and
references cited therein).
[0113] Binary vectors may replicate in both Agrobacterium species
and E. coli. They may contain a selectable marker and a poly-linker
region which to the left and right contains the border sequences of
the T-DNA of Agrobacterium rhizogenes or Agrobacterium tumefaciens.
Such vectors may be transformed directly into Agrobacterium
species. The Agrobacterium cell serving as the host cell has to
contain a vir-region on another plasmid. Additional T-DNA sequences
may also be contained in the Agrobacterium cell.
[0114] The Agrobacterium cell containing the DNA sequences to be
transferred into plant cells either on a binary vector or in the
form of a cointegrate between the intermediate vector and the T-DNA
region may then be used for transforming plant cells. Usually
either multicellular explants (e.g. leaf discs, stem segments,
roots), single cells (protoplasts) or cell suspensions are
cocultivated with Agrobacterium cells containing the DNA sequence
to be transferred into plant cells. The plant cells treated with
the Agrobacterium cells are then selected for the cotransferred
resistance marker (e.g. kanamycin) and subsequently regenerated to
intact plants. These regenerated plants will then be tested for the
presence of the DNA sequences to be transferred.
[0115] If the DNA is transferred by e.g. electroporation or
microinjection, no special requirements are needed to effect
transformation. Simple plasmids e.g. of the pUC series may be used
to transform plant cells. Regenerated transgenic plants may be
grown normally in a greenhouse or under other conditions. They
should display a new phenotype (e.g. production of new proteins)
due to the transfer of the foreign gene(s). The transgenic plants
may be crossed with other plants which may either be wild-type or
transgenic plants transformed with the same or another DNA
sequence. Seeds obtained from transgenic plants should be tested to
assure that the new genetic trait is inherited in a stable
Mendelian fashion.
[0116] See also Hiatt, Nature 344: 469-479, 1990; Edelbaum et al.,
J. Interferon Res. 12: 449-453, 1992; Sijmons et al., Bio/Tecnology
8: 217-221, 1990: and EP 255 378.
[0117] Uses
[0118] In the pharmaceutical composition of the invention, the
present polypeptide may be formulated by any of the established
methods of formulating pharmaceutical compositions, e.g. as
described in Remington's Pharmaceutical Sciences, 19 th.
edition,1995. The composition may be in a form suited for systemic
injection or infusion and may, as such, be formulated with sterile
water or an isotonic saline or glucose solution. The compositions
may be sterilized by conventional sterilization techniques which
are well known in the art. The resulting aqueous solutions may be
packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with the
sterile aqueous solution prior to administration. The composition
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as buffering
agents, tonicity adjusting agents and the like, for instance sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, etc.
[0119] The pharmaceutical composition of the present invention may
also be adapted for oral, nasal, transdermal, transepithelial or
rectal administration. The pharmaceutically acceptable carrier or
diluent employed in the composition may be any conventional solid
carrier. Examples of solid carriers are lactose, terra alba,
sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate
and stearic acid. Similarly, the carrier or diluent may include any
sustained release material known in the art, such as glyceryl
monostearate or glyceryl distearate, alone or mixed with a wax. For
oral administration, the composition may be tabletted, placed in a
hard gelatin capsule in powder or pellet form or it can be in the
form of a troche or lozenge. The amount of solid carrier will vary
widely but will usually be from about 25 mg to about 1 g. The
present polypeptide may also be placed in a soft gelatin capsule in
a liquid carrier such as syrup, peanut oil, olive oil or water.
[0120] The polypeptides of the invention are effective over a wide
dosage range. A typical dosage is in the range of from 0.05 to
about 1000 mg, preferably from about 0.1 to about 500 mg, and more
preferred from about 0.5 mg to about 200 mg per day administered in
one or more dosages such as 1 to 3 dosages. The exact dosage will
depend upon the frequency and mode of administration, the sex, age,
weight and general condition of the subject treated, the nature and
severity of the condition treated and any concomitant diseases to
be treated as well as other factors evident to those skilled in the
art.
[0121] The polypeptide of the invention is contemplated to be
advantageous for use in therapeutic applications within appetite
suppression or satiety induction, such as for the prophylaxis or
treatment of diseases or disorders associated with impaired
appetite regulation. Examples of such diseases or disorders are
obesity, type II diabetes and bulimia. The dosage of the
polypeptide administered to a patient will vary with the type and
severity of the condition to be treated, but is generally in the
range of 0.01-5.0 mg/kg body weight per day in one or more dosages
such as 1 to 3 dosages.
[0122] Furthermore, the polypeptide of the invention is
contemplated to be advantageous for treatment of autoimmune
disorders, inflammation, arthritis, type I diabetes, multiple
schlerosis, stroke, osteoporosis, septic shock, symptoms of
menopause, menstrual complications and Parkinson's disease.
[0123] The present invention is further illustrated by the
following representative examples which are, however, not intended
to limit the scope of the invention in any way. Fur further details
of the invention reference should also be made to Peter Kristensen
et al., "Hypothalamic CART: a novel anorectic peptide regulated by
leptin", Nature, in press, which is hereby incorporated by
reference.
EXAMPLES
Example 1
[0124] Isolation of CART
[0125] Total RNA from cultivated cells were prepared from primary
cell lines derived from the rat tumors MSL-A-AN and MSL-A-M3
(Madsen et al, Scand. J. Clin. Invest. Supplement 220: 27-36) by
the method of Chomczynski & Sacchi (1987). From this we made
poly-A mRNA using Pharmacia's "Quick Prep.RTM. micro" mRNA
purification kit. Double stranded cDNA was made using Clontech's
"cDNA clone I" synthesis kit. Eco RI adaptors CA6/CA7 (Ace et al.
(1994)) were added to the blunt ended cDNA's.
[0126] 50 ng of each cDNA was amplified using the CA6 primer. The
primer used for amplification of the MSL-A-M3 cDNA was biotinylated
(the driver). The amplified MSL-A-AN cDNA was cut with Eco RI (the
tracer).
[0127] 8 .mu.g of the biotinylated M3 cDNA was bound to 60 .mu.l of
DYNAL magnetic streptavidin beads, treated with NaOH, washed,
2.times.hybridization buffer was added, and the mixture was heated
to 68.degree. C. 0.5 .mu.g of the Eco RI cut AN cDNA was heated to
98.degree. C. and added to the tube with the magnetic beads. The
reaction tube was incubated at 68.degree. C. for 20 hours. The
magnetic beads with the bound cDNA and the tracer which had
hybridized to it was removed using a magnet.
[0128] This procedure was repeated three times, the last two with
10 .mu.g of driver DNA and 80 .mu.l of magnetic beads.
[0129] The remaining tracer DNA was purified on a Chromaspin 100
column from Clontech and cloned into an Eco RI cut vector
(pCR.TM.II, Invitrogen) and transformed into E. coli
(ElectroMax).
[0130] The cells were plated on LB plates with 100 .mu.g/ml
Ampicillin. The plates were replicated and the cells from the
replica were transferred to nylon filters (Hybond-N+, Amersham).
The filters were hybridized with radioactively labelled driver cDNA
and autoradiographs were made. The filters were then stripped for
the radioactive probe and thereafter the procedure was repeated
with a probe made from the tracer cDNA.
[0131] The autoradiographs were compared and spots that were only
present with the latter probe were identified and the corresponding
colonies were isolated.
[0132] DNA sequencing of the inserted DNA in one of these colonies
were identified as CART (Cocaine and Amphetamine Regulated
Transcript from rat brain (Douglass et al. (1995)). This transcript
codes for a protein (polypeptide) of 129 or 116 amino acids
(differential splicing of an in frame 39 bp intron). The
polypeptide seems to have a signal sequence in the amino terminal
end, and the secreted part contains several dibasic amino acid
pairs which could be "pro hormone" processing sites.
Example 2
[0133] Cloning of Rat CART
[0134] In order to clone the whole coding region of the CART gene
primers were made that overlaps with the start codon and with the
stop codon, respectivly.
[0135] MHJ4754: 5'-AAAAAGGATCCACCATGGAGAGCTCCCGCC-3'
[0136] Bold: Bam HI site for cloning. Underlined: ATG start
codon.
[0137] MHJ4753: 5'-AAAAAAGCTTCACAAGCACTTCAAGAGGAAA-3'
[0138] Bold: Hin dIII site for cloning. Underlined: TGA stop codon,
opposite strand.
[0139] As template for the PCR cloning we used the same double
stranded cDNA preparation as described in Example 1 (from
MSL-A-AN).
4 The PCR reaction, 25 cycles: 60 sec 94.degree. C. 30 sec
52.degree. C. 60 sec 72.degree. C.
[0140] Two bands appeared when the reaction mix was run on a 2%
agarose gel corresponding to the two splice variants mentioned in
Douglass et al. (1995).
[0141] Each of the two bands were isolated, cut with Bam HI and Hin
dIII, and cloned into Bam HI-Hin dIII cut pSX221 (fragments
A,B,C,D, and E ligated into pSX191, WO 92/11357) giving rise to
pSX592 and pSX593 (short and long form, respectively) corresponding
to SEQ ID Nos. 2 and 1, respectively (see FIG. 1).
Example 3
[0142] Expression of Rat CART in E. coli I
[0143] In order to express CART in E. coli three constructs were
made where different forms of CART were fused to Glutathione
S-transferase using the pGEX system from Pharmacia P-L
Biochemicals.
[0144] The different forms of CART, full length of both splice
variants (starting with Gln-Glu-Asp) and the form starting with
Ile-Pro-Ile (Spiess et al. (1981)) were amplified using PCR primers
that add a Bam HI site (bold) and the four triplets that codes for
the Factor Xa protease recognition site Ile-Glu-Gly-Arg in the
5'-end (N-terminus). As 3'-end primer we used the same as in
Example 2 (MHJ4753). As templates were used the plasmids pSX592 and
pSX593 described in Example 2.
5 MHJ4885: 5'-AAAAAGGATCC ATC GAA GGT CGT CAG GAG GAT GCC GAG
CTG-3' Ile Glu Gly Arg Gln Glu Asp Ala Ser Leu MHJ4880:
5'-AAAAAGGATCC ATC GAA GGT CGT ATT CCG ATC TAT GAG AAG A-3' Ile Glu
Gly Arg Ile Pro Ile Tyr Glu Lys
[0145]
6 The PCR reaction, 25 cycles: 60 sec 94.degree. C. 30 sec
55.degree. C. 60 sec 72.degree. C.
[0146] The reaction mixtures were cut with Hin dIII, filled out
with Klenow polymerase, and then cut with Bam HI. They were then
run on a 2% agarose gel and the bands corresponding to the three
variants were isolated and cloned into pGEX-2T (cut with Eco RI,
filled out, and then cut with Bam HI) giving rise to pSX600
(IPI-CART) corresponding to SEQ ID No. 4, pSX 601 (short form)
corresponding to SEQ ID No. 2, and pSX605 (long form) corresponding
to SEQ ID No. 1 (see FIG. 2).
Example 4
[0147] Expression of Rat CART in E. coli II
[0148] In order to express CART in E. coli three constructs were
made where different forms of CART were fused to Thioredoxin using
the ThioFusion.TM. Expression System from Invitrogen
Corporation.
[0149] The different forms of CART, full length of both splice
variants (starting with Gln-Glu-Asp) and the form starting with
Ile-Pro-Ile (Spiess et al. (1981)) were amplified using PCR primers
that add a Bam HI site (bold) and the four triplets that codes for
the Factor Xa protease recognition site Ile-Glu-Gly-Arg in the
5'-end (N-terminus) and a Hin dIII (bold) site in the 3'-end. As
templates were used the pGEX fusion constructs described in Example
3.
7 MHJ5141: 540 -AAAAAGGATCCG ATC GAA GGT CGT GAG GAG GAT-3' Ile Glu
Gly Arg Gln Glu Asp MHJ5140: 540 -AAAAAGGATCCG ATG GAA GGT CGT ATT
CGG ATC-3' Ile Glu Gly Arg Ile Pro Ile MHJ5142:
5'-AAAAAGTCGATAAGCTTCACAAGCACTTCAAGAGGA-3'
[0150] Bold: Hin dIII Underlined: Stop codon on opposite strand
8 The PCR reaction, 25 cycles: 60 sec 94.degree. C. 30 sec
52.degree. C. 60 sec 72.degree. C.
[0151] The reaction mixtures were cut with Hin dIII, filled out
with Klenow polymerase and then cut with Bam HI. They were then run
on a 2% agarose gel and the bands corresponding to the three
variants were isolated and cloned into pTrxFus (cut with Sal I,
filled out, and then cut with Bam HI) giving rise to pSX610 (long
form) corresponding to SEQ ID No. 1, pSX611 (short form)
corresponding to SEQ ID No. 2, and pSX612 (IPI-CART) corresponding
to SEQ ID No. 4 (see FIG. 3).
[0152] The plasmids were transformed into E. Coli G1724
(Invitrogen) and the the resulting strains were cultivated
according to the manual for the ThioFusion.TM. Expression System
kit.
[0153] The fusion proteins were purified according to the
instruction manual for ThioBond.TM. Resin (Invitrogen Corporation).
The purified fusion proteins were then treated with the
endoproteinase Factor Xa (Boehringer Mannheim). Ratio Factor
Xa/Fusion protein=1/800. Incubation: 4.degree. C., 16 hours.
Example 5
[0154] Expression of Rat CART in S. cerevisiae
[0155] The Yeast-E.coli shuttle vector used in the following
example (pAK405) contains a heterologous protein expression
cassette, which includes a DNA sequence encoding a modified
MF.alpha.1 leader sequence (with a NcoI site added in the 3'-end)
followed by the heterologous polypeptide in question operably
placed in between the TPI promoter and the TPI terminator of S.
cerevisiae in a POT plasmid (Kjeldsen et al., Gene 170:107-112,
1996).
[0156] Two primers CART1 and CART2 were constructed. These allow a
PCR product to be made that furnishes the DNA sequence encoding
either the short or the long form of full length CART with a 5'
NcoI site and a 3' XbaI site allowing insertion into the
yeast-E.coli shuttle vector pAK405.
9 NcoI site
.about..about..about..about..about..about..about..about..about.
CART1: 5'-ACG GAG GAG CCC ATG GCT AAG AGA CAG GAG GAT GCC GAG CTG
CAG C-3' XbaI site
.about..about..about..about..about..about..about..about..about.
CART2: 5'-CTT AAC GGC TTC TAG ATC ACA AGC ACT TCA AGA GG-3'
[0157] The following Polymerase Chain Reaction (PCR) was performed
using the Pwo DNA polymerase (Boehringer Mannheim) according to the
manufacturers instructions.
[0158] 5 .mu.l primer CART1 (100 pmol)
[0159] 5 .mu.l primer CART2 (100 pmol)
[0160] 10 .mu.l 10.times.PCR buffer+MgSO.sub.4
[0161] 8 .mu.l dNTP mix
[0162] 0.5 .mu.l Pwo enzyme
[0163] 1 .mu.l pSX592 or pSX593 plasmid as template (0.2 .mu.g
DNA)
[0164] 70.5 .mu.l H.sub.2O
[0165] A total of 16 cycles was performed. One cycle was as
follows: 45 sec at 94.degree. C., 1 min at 42.degree. C., and 1.5
min at 72.degree. C.
[0166] The resulting PCR products were cut with restriction enzymes
NcoI and XbaI and ligated with the BstXI/XbaI fragment and the
BstXI/NcoI fragment of pAK405. BstXI, NcoI and XbaI cutat positions
701, 1419 and 1616 of pAK405, respectively. The construction and
DNA sequence of the resulting heterologous expression cassettes are
shown in FIGS. 4 and 5, respectively.
[0167] The resulting plasmids pEA182 (short form of CART) and
pEA183 (long form of CART) were transformed into S. cerevisia
strain ME1487 (MAT.alpha. .DELTA.yap3::URA3 pep4-3 .DELTA.tpi::LEU2
leu2 HIS4 .DELTA.URA3, described in patent application DK 0749/96).
Transformants were selected by glucose utilization as a carbon
source in YPD plates (1% w/v yeast extract, 2% w/v peptone, 2%
glucose, 2% agar). yEA182 corresponding to SEQ ID No. 2 and yEA183
corresponding to SEQ ID No. 1 are the transformants obtained from
the plasmids pEA182 and pEA183, respectively.
[0168] A similar construct was made which produces IPI-CART: pSX630
corresponding to SEQ ID No. 4.
[0169] Transformants were cultivated in YPD liquid medium at
30.degree. C. for 3 days with shaking at 200 rpm. Culture
supernatants were obtained after centrifugation and supernatants
were analysed for CART related material.
[0170] Human CART(1-89) corresponding to SEQ ID No. 3 differs from
the rat form by having a valine residue in position 42 in stead of
a isoleucine residue. Human CART(1-89) may be prepared in analogy
with the above examples starting from a human tissue or simply by
substituting valine for isoleucine in position 42 of rat CART(1-89)
according to methods well-known to a person skilled in the art.
Example 6
[0171] Preparation of Rat CART(68-102, Long). SEQ ID No. 9
[0172] Ten mg of rat CART(54-102, long), SEQ ID No.6, prepared as
described in Example 7, was dissolved in 2 ml of 70% (v/v) formic
acid. A crystal corresponding to approx. 1 mg of cyanogenebromide
was added to the dissolved peptide and the mixture was allowed to
stand dark at room temperature for 16 hours. The generated CART
fragment, CART(68-102, long) was purified by preparative HPLC as
described in Example 7.
Example 7
[0173] E.Coli Construction
[0174] The thioredoxin-CART short form fusion protein was isolated
from 2 litres of E.Coli fermentation broth and subjected to FXa
cleavage.
[0175] This digest mixture was analysed by HPLC (FIG. 6). Fractions
corresponding to the two main peaks (Fr. 15 and Fr. 28, FIG. 6)
were subjected to sequence and mass spectrometry analysis:
10 Fr. No. Sequence found Mass found Theoretical mass 15 ALDIYSAVDD
. . . 8882.8 8882.4 28 SDKIIHLTDD . . . 13529.0 13529.5
[0176] The pept
[0177] ide found in Fraction 15 is identical to rat CART(10-89)
corresponding to SEQ ID No. 10, whereas the peptide in Fraction 28
is the thioredoxin split product with the C-terminal sequence of .
. . IEGR. The small peptide fragment, CART(1-9), was not identified
in the digest.
[0178] From 2.1 of fermentation broth the total of 4.0 mg of
CART(10-89) was isolated.
[0179] Human CART(10-89) corresponding to SEQ ID No. 11 differs
from the rat form by having a valine residue in position 33 in
stead of a isoleucine residue. Human CART(10-89) may be prepared in
analogy with the above example starting from a human tissue or
simply by substituting valine for isoleucine in position 33 of rat
CART(10-89) according to methods well-known to a person skilled in
the art.
[0180] Yeast Construction
[0181] The fermentation broth from the 5 litres yeast fermentation
(yEA183, long form) was analysed by HPLC (FIG. 7). A series of
expression products was seen in this analysis.
[0182] Preliminary sequence analysis indicated that several of the
peptides eluting at a retention time between 20 and 30 min. were
fragments of the mature full length CART molecule. The total amount
of CART related products in the fermentation broth was approx. 250
mg/litre.
[0183] The CART fragments from 4.25 litres of fermentation broth
were separated using the following method:
[0184] The fermentation broth (pH=4.6, .LAMBDA.=8 mS) was adjusted
to pH=5.0 and diluted with 25 litres of water (resulting
.LAMBDA.=1.3 mS) and pumped (500 ml/h) onto a SP-Sepharose column
(5.times.15 cm) previously equilibrated with 50 mM HAc at pH=5.0.
The column was eluted with a linear gradient between 1500 ml of 50
mM HAc and 1500 ml of 50 mM HAc containing 1.0 M NaCl. Fractions of
10 ml were collected and analysed for the content of CART fragments
by analytical HPLC. The chromatogram from the ion exchange
chromatography is shown in FIG. 8. Three pools (A, B and C, see
FIG. 8) were generated on the basis of the analytical HPLC analysis
of the individual fractions. Each of these pools, representing a
well defined CART fragment, were further purified by preparative
HPLC. The individual pools (120-150 ml) were pumped on a Vydac
214TP1022 (100 ml) reverse phase C4 HPLC column previously
equilibrated with 0. 1% TFA. The column was washed with 100 ml 0.1%
TFA and eluted with a linear gradient from 0 to 70% MeCN in 0.1%
TFA at a flow rate of 3 ml/min. The individual fractions from the 3
preparative HPLC purifications were analysed by analytical HPLC and
the 3 individual CART fragments were isolated from these fractions
by lyophilisation.
[0185] Characterisation of the Isolated CART Fragments From the
Yeast Fermentation
[0186] The purity of the 3 isolated CART fragments are shown in
FIGS. 9, 10, and 11, respectively. The structure of the 3 purified
CART fragments were determined by amino acid sequencing and
MALDI-TOF mass spectrometry. The following results were found:
11 Pool Mass Theoretical No. Sequence found found mass Identity A
YGQVPM . . . 4389.9 4387.1 CART(62-102) B KYGQVP . . . 4516.5
4515.3 CART(61-102) C RIPIYEKKY . . . 5418.0 5415.4
CART(54-102)
[0187] The total yields of the purified rat CART fragments from
4.25 litres of fermentation broth were:
12 Pool No. Identity Total Yield A CART(62-102) 33 mg B
CART(61-102) 200 mg C CART(54-102) 280 mg
[0188] CART(62-102) corresponds to SEQ ID No. 8, CART(61-102)
corresponds to SEQ ID No. 7 and CART(54-102) corresponds to SEQ ID
No. 6.
[0189] The human CART(62-102) and CART(61-102) fragments are
identical to the rat fragments. Human CART(54-102) differs from the
rat fragment by having a valine residue in position 2 in stead of a
isoleucine residue. Human CART(54-102) may be prepared using the
same method as described for rat CART(54-102) starting from a human
tissue or simply by substituting valine for isoleucine in position
2 of rat CART(54-102) according to methods well-known to a person
skilled in the art.
Example 8
[0190] The Disulphide Bond Configuration in Rat CART(62-102) SEQ ID
No. 8
[0191] The C-terminal part of the CART molecule contains 6 cysteine
residues:
13 Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-
Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-
Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-
Leu-Leu-Lys-Cys-Leu
[0192] In principle these 6 cysteine residues can exist in
5.times.3.times.1=15 possible arrangements to form 3 disulphide
bonds. The present series of experiments were carried out in order
to elucidate, which of the 15 possible arrangements was present in
the CART molecule.
[0193] The CART fragment (residues 62-102) prepared as described in
the preceding example was digested with Armillaria Mellea protease,
which cleaves specifically on the N-terminal side of lysine
residues. The fragments generated were separated by HPLC and
subjected to mass spectrometry and amino acid sequence analyses.
From mass spectrometry and sequence analysis it could be deduced
that the following two fragments were generated by the Armillaria
Mellea protease digestion:
14 Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg
Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu
Lys-Cys-Leu Lys-Gly-Ala-Arg-Ile-Gly
[0194] The first of these fragments is a 3 chained molecule still
held together by the 3 disulphide bonds. This molecule was
subjected to digestion with Pseudomonas fragi endoproteinase Asp-N,
which cleaves specifically on the N-terminal side of aspartic acid
residues. From this digest the following two fragments could be
isolated:
15 Tyr-Gly-Gln-Val-Pro-Met-Cys Lys-Leu-Cys
Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cy-
s-Asn-Ser-Phe-Leu- Leu Lys-Cys-Leu
[0195] The first of these fragments is a two chained molecule held
together by a single disulphide bond. Thus, cysteine residue I and
III of the original molecule must be linked.
[0196] The second of these fragments, containing cysteine residue
II, IV, V and VI, is a 3 chained molecule linked by 2 disulphide
bonds. This molecule was subjected to trypsin digestion and the
following two fragments were generated:
16 Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg
Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu Asp-Cys-Pro-Arg Lys-Cys-Leu
[0197] From these results it is clear that Cys-II and Cys-V are
linked and that Cys-IV and Cys-VI are linked.
[0198] From the above results the entire primary and secondary
structure of the C-terminal part of the CART molecule can be
deduced showing that the disulphide bond exists in a I-III, II-V
and IV-VI configuration (see FIG. 12).
Example 9
[0199] Test Method for Measuring Appetite Suppression in Mice
[0200] Mice were deprived of their normal feed for two days and
given free access to a solution of nutritionally complete infant
formula milk (Complan.RTM.) for the first day, after which food
deprivation was complete for the last day before testing. After one
day of food deprivation, mice were injected
intra-cerebroventricularly (ICV) in the lateral ventricle with 10
microlitres of a solution containing the test substance. Thirty
minutes after injection, mice were individually placed in a
15.times.15.times.15 cm test box with a stainless steel grid floor
and a glass drinking tube which projected into the box. The
drinking tube was connected to a reservoir containing the formula
milk solution, and the interior of the drinking tube contained an
electrode enabling the detection of drinking contacts with the
solution by measuring the flow of a weak (unnoticeable) electric
current through mice by means of an electronic apparatus connected
to the drinking tube electrode and the stainless steel grid floor.
Consumption of the milk solution was measured over a 10 minutes
period by electronically recording the total amount of contact with
the milk solution during the test session. The degree of appetite
suppression produced by a given test substance was determined by
statistical comparison of the duration of milk consumption by
control (vehicle treated) mice with that of mice treated with a
test substance. The degree of appetite suppression in a treated
group of mice was expressed as percent reduction of consumption
relative to the mean of the control group's response.
Example 10
[0201] Test for Appetite Suppression in Mice by Recombinant Rat
CART(10-89. Short), SEQ ID No. 10
[0202] Mice were tested for appetite suppression as described in
Example 9 after treatment with 1-20 micrograms of a test substance
consisting of recombinant rat CART(10-89) dissolved in phosphate
buffered saline. Intra-cerebroventricular injections of the test
substance produced statistically significant suppression of milk
consumption.
17 Dose (micrograms) 1 2 5 10 20 % Feeding Suppression 41% 70% 71%
59% 78%
Example 11
[0203] Test for Appetite Suppression in Mice by Recombinant Rat
CART(54-102, Long), SEQ ID No. 6
[0204] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.5-10 micrograms of a test
substance consisting of recombinant rat CART(54-102) dissolved in
phosphate buffered saline. Intra-cerebroventricular injections of
the test substance produced Statistically significant suppression
of milk consumption.
18 Dose (micrograms) 0.5 1 2 5 10 % Feeding Suppression 13% 71%
100% 100% 100%
Example 12
[0205] Test for Appetite Suppression in Mice by Recombinant Rat
CART(61-102. Long). SEQ ID No. 7
[0206] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.5-10 micrograms of a test
substance consisting of recombinant rat CART(61-102) dissolved in
phosphate buffered saline. Intra-cerebroventricular injections of
the test substance produced statistically significant suppression
of milk consumption.
19 Dose (micrograms) 0.5 1 2 5 10 % Feeding Suppression 74% 80% 78%
100% 100%
Example 13
[0207] Test for Appetite Suppression in Mice by Recombinant Rat
CART(62-102, Long), SEQ ID No. 8
[0208] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.5-10 micrograms of a test
substance consisting of recombinant rat CART(62-102) dissolved in
phosphate buffered saline. Intra-cerebroventricular injections of
the test substance produced statistically significant suppression
of milk consumption.
20 Dose (micrograms) 0.5 1 2 5 10 % Feeding Suppression 30% 55% 60%
89% 100%
Example 14
[0209] Effect of Fasting on the Expression of CART mRNA
[0210] Rat brain tissue from three different groups of animals (6
animals in each group): normal control, fasted for 48 hours and
fasted for 48 hours and re-fed for 3 hours. Cryostat sections Were
cut and three sections were used for in situ hybridisation with
35-S labelled anti-sense CART RNA. Additional sections were
included with a similar amount of 35-S labelled sense RNA probe.
Slides were exposed on one Betamax hyperfilm for 7 days. The images
were digitized and analysed using the NIH Image software (treatment
blinded to the observer). An empirically determined gray level was
used to set the threshold on all images after exclusion of those
with bad morphology or bad representation of the area in question
(indicated by lines on the figure). The average gray scale value of
all pixels above this level within the area of interest (PVN or
Nucleus Arcuatus (in both the frontal section and at eminentia
mediana)) was then measured and multiplied by the size of the area
of interest. A mean for each animal was then determined and the
standard deviation indicated represents the spreading between
animals.
[0211] These results show that CART mRNA is regulated in a manner
inverse to that of NPY thus indicating the presence of
neurotransmitter mode action for CART involving a receptor
mediating a satiety stimulus, presumably along the
arcuate--paraventricular nucleus pathway (see FIG. 13).
Example 15
[0212] Low Expression of CART mRNA in Arcuate Nucleus of Obese
Zucker Rats
[0213] Rat brain tissue was obtained from two groups of Zucker rats
(6 animals in each group): 25 fa/fa and fa/+. Cryostat sections
were cut and three sections were used for in situ hybridisation
with 35-S labelled anti-sense CART RNA (rCART5A cDNA (Eco47-HindIII
fragment from bp Nos. 226-411)). Post-hybridisation washings were
performed at 57.degree. C. and 62.degree. C. in 50% formamide.
Additional sections were included with a similar amount of 35-S
labelled sense RNA probe and these showed no signal. Slides were
exposed on one Betamax hyperfilm for 12 days. The images were
digitized to 256 grey levels and analysed using the NIH Image
software (treatment blinded to the observer). An empirically
determined gray level (100) was used to set the threshold on all
images after exclusion of those with bad morphology or bad
representation of the area in question (three sections) and one set
of slides (Nos. 25-27) as one animal due to an error was
represented twice (and one missing). The average gray scale value
of all pixels above the arcuate nucleus was then measured. A mean
for each animal was then determined and the product of the area and
mean calculated. These results show that CART mRNA is regulated in
a manner inverse to that of NPY thus indicating the presence of
neurotransmitter mode action for CART involving a receptor
mediating a sateity stimulus, presumably along the
arcuate--paraventricular nucleus pathway. Furthermore, the strong
decrease in CART expression in the obese Zucker rat deficient in
leptin signalling strongly implicates CART mediated neuronal
signalling in a sateity mediating pathway in the hypothalamus (see
FIG. 14).
Example 16
[0214] Preparation of Rat CART(55-102). SEQ ID No. 4
[0215] Plasmid pSX637, encoding Glu-Glu-Ile-Asp-CART(55-102), was
constructed by the use of the PCR technique "Splicing by Overlap
Extension" (Horton et al., Gene 77:61-68, 1989) and the product was
inserted into pAK405 (Example 5). The resulting expression plasmid
(pSX637) is shown in FIG. 15. As can be seen from this figure a
sequence of: Lys-Glu-Leu-Glu has been placed between the
.alpha.-leader and Kex2 site in order to optimise processing
(Kjeldsen et al., Gene 170:107-112, 1996).
[0216] Plasmid pSX637 was transformed into Saccharomyces.
cerevisiae strains ME1487 (MAT.alpha. .DELTA.yap3::URA3
.DELTA.tpi::LEU2 pep4-3 .DELTA.ura3 leu2) and ME1719
(MAT.alpha./MAT.alpha. .DELTA.yap3::URA3/.DELTA.yap3::URA3
.DELTA.tpi::LEU2/.DELTA.tpi::LEU2 pep4-3/pep4-3
.DELTA.ura3/.DELTA.ura3 leu2/leu2), respectively. Host cells were
cultured in YPGGE medium (1% (w/v) yeast extract, 2% (w/v) peptone,
2% (w/v) galactose, 2% (v/v) glycerol and 1 % (v/v) ethanol) to
OD.sub.600 nm of 0.2. Transformation was made by using a standard
protoplast method. Transformant YES1789, was obtained containing
the EEID-CART expressing plasmid after transformants were selected
on minimal plates containing glucose.
[0217] Fermentation of EEID-CART(55-102) (yeast strain: YES1789)
was carried out in 6 L stainless steel fermentor from Chemap A/B,
Switzerland equipped with bottom stirrer and in situ steam
sterilization. The medium was composed essentially as previously
described (Thim et al., FEBS Lett. 318:345-352, 1993) and a
starting volume of 4.0 L was chosen. Ammonia was added to adjust pH
throughout the fermentation to 4.9 and the temperature was kept
constant at 30.degree. C. with steam/cooling water. Dissolved
oxygen was kept above 50% saturation by frequent adjustment of the
stirrer speed. The inoculum was from a YPD (Yeast-extract Peptone
Dextrose) culture (2 days, 30.degree. C.). Glucose (1250 g) was
dissolved in water to a volume of 2 L, sterilised separately in an
autoclave (30 min, 121 .degree. C.), and added with a constant rate
of 30 g/h over the first 24 hours. The rate was increased to 60 g/h
over the next 24 hours. After 48 hours of cultivation the broth was
harvested.
[0218] The EEID-CART(55-102) broth was adjusted to pH 11 with 3 N
NaOH and kept at 25.degree. C. for 30 min before centrifugation as
above. The supernatant was adjusted to pH 9.7 to protect against
proteolysis before the purification was initiated. The dry biomass
in the two fermentations was 73.6 g/L. The weight of the total
fermentation broth was 5706 g. The fermentation supernatant (4.6 L)
from yeast strain YES1789 expressing Glu-Glu-Ile-Asp-CART(55-102)
was dialysed against 60 L of water at 4.degree. C. for 96 h. The pH
was adjusted to 4.3 and the solution was pumped onto a SP-Sepharose
(Pharmacia) column (5.times.15 cm) with a flow rate of 300 mL/h.
Prior to the application the column was equilibrated with 50 mM HAc
buffer pH 4.25. The column was washed with 3 L of 50 mM HAc buffer
pH 4.25. EEID-CART(55-102) was eluted from the column by a linear
gradient between 1.5 L of 50 mM HAc buffer pH 4.25 and 1.5 L of 50
mM HAc buffer pH 4.25 containing 1M NaCl. Fractions (10 mL) were
collected at a flow rate of 100 mL/h and the absorbance was
measured at 280 nm. The EEID-CART(55-102) molecule eluted at 0.5M
of NaCl and fractions containing the peptide were dialysed against
25 L of 50 mM HAc buffer pH 4.5 at 4.degree. C. for 96 h. L-Cystein
was added to the solution (560 mL) to give a final concentration of
1 mM, and 4.5 mL dipeptidylaminopeptidase-1 ( DAP-1, Cathepsin C
from chicken liver, EC 3.4.14.1, Unizyme Laboratories) was added.
The resulting concentration of DAP-1 was 20 units/mL. The digestion
of EEID-CART(55-102) was carried out at 37.degree. C. and aliquots
were analysed by HPLC each half hour. After incubation for 4.5 h
more than 98% of the precursor was converted to CART(55-102). The
digestion mixture was adjusted to pH 4.25 and pumped (60,mL/h) onto
a SP-Sepharose (Pharmacia) column (5.times.8 cm) previously
equilibrated with 50 mM HAc buffer pH 4.25. The column was washed
with 1.6 L of equilibration buffer and CART(55-102) was eluted with
a linear gradient between 1 L of 50 mM HAc buffer pH 4.25 and 1 L
of 50 mM HAc buffer pH 4.25 containing 1.2 M NaCl, at a flow rate
of 100 mL/h. The absorbance at 280 nm was recorded and fractions of
10 mL were collected. The CART(55-102) molecule eluted at 0.67 M of
NaCl and fractions containing the peptide were pooled. The solution
was divided into 5 equal portions. Each portion was pumped on a
Vydac 214TP1022 reverse phase C4 preparative HPLC column
(2.2.times.25 cm) previously equilibrated with 0.1% (v/v) TFA. The
column was washed with 100 mL 0.1% (v/v) TFA and eluted with a
linear gradient from 0 to 70% (v/v) acetonitrile in 0.1% (v/v) TFA
at a flow rate of 3 mL/min. The CART(55-102) containing fractions
from the 5 preparative HPLC purifications were pooled and the
peptide was isolated from these fractions by lyophilisation. The
total yield of CART(55-102) from 4.6 L of fermentation supernatant
was 705 mg.
[0219] N-terminal amino acid sequences were determined by automated
Edman degradations using an Applied Biosystem Model 494 Protein
Sequencer essentially as described by the manufacturer. The
N-terminal sequence of the purified CART(55-102) was found to
be:
[0220] IPIYEKKYGQ . . .
[0221] Mass spectrometric analysis on the isolated CART(55-102) was
performed on a Voyager RP MALDI-TOF instrument (Perseptive
Biosystems Inc., Framingham, Mass.) equipped with a nitrogen laser
(337 nm). The instrument was operated in linear mode with delayed
extraction, and the accelerating voltage in the ion source was 25
kV.
[0222] Sample preparation was done as follows: 1 .mu.L sample
solution was mixed with 10 .mu.L matrix solution
(alpha-cyano-4-hydroxy-cinnamic acid dissolved in a 5:4:1 (v/v/v)
mixture of acetonitrile:water:3% (v/v) TFA) and 1 .mu.L was
deposited on the sample plate and allowed to dry. Calibration was
performed using external standards and the accuracy of the mass
determinations is within 0.1%. The mass found for the isolated
CART(55-102) was 5257.1 as compared to a calculated mass of
5255.5.
Example 17
[0223] Test for Appetite Suppression in Mice by Recombinant Rat
CART(55-102, Long), SEQ ID No. 4
[0224] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.1-1.0 micrograms of a test
substance consisting of recombinant CART(55-102) dissolved in
phosphate buffered saline. Intra-cerebroventricular injections of
the test substance produced a statistically significant suppression
of milk consumption.
21 Dose (micrograms) 0.1 0.2 0.5 1.0 % Feeding Suppression 38% 61%
98% 99%
[0225] The human CART(55-102) peptide corresponding to SEQ ID No. 5
differs from the rat form by having a valine residue in stead of a
isoleucine residue in position 1. It may be prepared using the same
method as described for the rat form starting from a human tissue
or simply by substituting valine for isoleucine in position 1 of
the rat form.
Example 18
[0226] Test for Appetite Suppression in Mice by Fragmented
Recombinant Rat CART(55-102, Long), SEQ ID No. 4
[0227] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.1-2.0 .mu.g of a test substance
consisting of fragmented recombinant rat CART(55-102) (fragmented
by trypsin and endopeptidase Asp-N) dissolved in phosphate buffered
saline. Intra-cerebroventricular injections of the test substance
did not produce statistically significant suppression of milk
consumption at any dose tested (see table).
22 Dose (micrograms) 0.1 0.2 0.5 1 2 % Feeding Suppression 1% 0% 3%
0% 0%
Example 19
[0228] Test for Appetite Suppression in Mice by Recombinant Rat
CART(55-102, Long), SEQ ID No. 4 With a Disrupted Secondary
Structure
[0229] Mice were tested for appetite suppression as described in
Example 9 after treatment with 0.1-2.0 .mu.g of a test substance
consisting of recombinant rat CART(55-102) with a disrupted
secondary structure (reduced and pyridylated) dissolved in
phosphate buffered saline. Intra-cerebroventricular injections of
the test substance did not produce statistically significant
suppression of milk consumption at any dose tested (see table).
23 Dose (micrograms) 0.1 0.2 0.5 1 2 % Feeding Suppression 9% 12%
0% 26% 18%
Example 20
[0230] Test Method for Measuring Appetite Suppression After
Intra-Cerebral Injection of a Test Substance in Rats
[0231] Male Wistar rats were implanted with a guide cannula in the
lateral cerebral ventricle and allowed to recover for 4-8 days
before screening for functional cannulae. This was accomplished by
injection of 5 .mu.g of porcine neuropeptide Y (NPY) , which
stimulates feeding with intra-cerebral administration. Animals not
responding to NPY were discarded, and the remaining rats were
sorted into response-equivalent groups of 5-6 rats each. To test
the appetite suppressing effects of compounds, the rats were first
food deprived for 24 hours and then received a 5 .mu.l
intra-cerebroventricular injection of a test substance dissolved in
phosphate buffered saline (PBS). A control group injected with 5
.mu.l of PBS provided reference data for each experiment.
Consumption of a special test food (a mash made from a 2:1 mixture
of water and dry standard chow) was measured for one hour following
the injection. The degree of appetite suppression-produced by a
given test substance was determined by statistical comparison of
the amount of food consumed by control rats (vehicle treated) with
that of rats treated with a test substance. The degree of feeding
suppression in a group of rats was expressed as percent reduction
of consumption relative to the mean amount consumed by the control
group.
Example 21
[0232] Test for Appetite Suppression in Rats by Recombinant Rat
CART(55-102, Long), SEQ ID No. 4
[0233] Rats were tested for appetite suppression as described in
Example 20 after treatment with 1 .mu.g of a test substance
consisting of recombinant rat CART(55-102) dissolved in phosphate
buffered saline. Intra-cerebroventricular injection of 1 .mu.g of
the test substance produced a statistically significant 52%
suppression of food consumption.
Example 22
[0234] Test for Appetite Suppression in Rats by Recombinant Rat
CART(55-102, Long), SEQ ID No. 4
[0235] Rats were tested for appetite suppression as described in
Example 20 after treatment with 0.1-3.0 .mu.g of a test substance
consisting of recombinant rat CART(55-102) dissolved in phosphate
buffered saline. Intra-cerebroventricular injection of 0.3 .mu.g
and 3.0 .mu.g of the test substance produced statistically
significant suppression of food consumption (see table).
24 Dose (micrograms) 0.1 0.3 1 3 % Feeding Suppression 17% 45% 19%
63%
[0236]
Sequence CWU 1
1
* * * * *