U.S. patent application number 10/956724 was filed with the patent office on 2005-06-02 for hydrophobically-modified protein compositions and methods.
This patent application is currently assigned to Biogen Idec Inc.. Invention is credited to Baker, Darren P., Galdes, Alphonse, Garber, Ellen A., Pepinsky, R. Blake, Petter, Russell C., Porter, Jeffery, Strauch, Kathryn L., Taylor, Frederick R., Weinreb, Paul H., Wen, Dingyi, Williams, Kevin P., Zeng, Chenhui.
Application Number | 20050119181 10/956724 |
Document ID | / |
Family ID | 34624038 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050119181 |
Kind Code |
A1 |
Pepinsky, R. Blake ; et
al. |
June 2, 2005 |
Hydrophobically-modified protein compositions and methods
Abstract
Hydrophobically-modified proteins and methods of making them are
described. A hydrophobic moiety is attached to a surface amino acid
residue of the protein. The hydrophobic moiety can be a lipid or a
peptide. Alternatively, the protein can be derivatized by a wide
variety of chemical reactions that append a hydrophobic structure
to the protein. The preferred protein is of mammalian origin and is
selected from the group consisting of Sonic, Indian, and Desert
hedgehog. The hydrophobic moiety is used as a convenient tether to
which may be attached a vesicle such as a cell membrane, liposome,
or micelle.
Inventors: |
Pepinsky, R. Blake;
(Arlington, MA) ; Galdes, Alphonse; (Arlington,
MA) ; Garber, Ellen A.; (Cambridge, MA) ;
Baker, Darren P.; (Hingham, MA) ; Porter,
Jeffery; (Lexington, MA) ; Taylor, Frederick R.;
(Milton, MA) ; Williams, Kevin P.; (Chapel Hill,
NC) ; Petter, Russell C.; (Stow, MA) ;
Strauch, Kathryn L.; (Bedford, MA) ; Weinreb, Paul
H.; (Methuen, MA) ; Wen, Dingyi; (Waltham,
MA) ; Zeng, Chenhui; (Sudbury, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Biogen Idec Inc.
Cambridge
MA
Curis, Inc.
Cambridge
MA
|
Family ID: |
34624038 |
Appl. No.: |
10/956724 |
Filed: |
October 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10956724 |
Oct 1, 2004 |
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09579680 |
May 26, 2000 |
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09579680 |
May 26, 2000 |
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PCT/US98/25676 |
Dec 3, 1998 |
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60067423 |
Dec 3, 1997 |
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60099800 |
Sep 10, 1998 |
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60089685 |
Jun 17, 1998 |
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60078935 |
Mar 20, 1998 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 514/19.1; 514/3.8; 514/5.9; 514/7.7;
514/8.2; 530/409; 536/23.5 |
Current CPC
Class: |
A61K 38/1709 20130101;
C07K 2319/00 20130101; C07K 14/46 20130101 |
Class at
Publication: |
514/012 ;
530/350; 435/069.1; 435/320.1; 435/325; 536/023.5; 530/409 |
International
Class: |
A61K 038/17; C07K
014/47 |
Claims
What is claimed is:
1. An isolated, protein comprising an N-terminal amino acid and a
C-terminal amino acid, wherein the protein is selected from the
group consisting of: (a) a protein with an N-terminal cysteine that
is appended with at least one hydrophobic moiety; (b) a protein
with an N-terminal amino acid that is not a cysteine appended with
at least one hydrophobic moiety; and (c) a protein with at least
one hydrophobic moiety substituted for the N-terminal amino
acid.
2. The protein of claim 1, wherein the hydrophobic moiety is a
peptide comprising at least one hydrophobic amino acid.
3. The protein of claim 1, wherein the hydrophobic moiety is a
lipid.
4. The protein of claim 1, wherein the protein further comprises a
hydrophobic moiety substituted for, or appended to, the C-terminal
amino acid.
5. The protein of claim 1, wherein the protein is an extracellular
signaling protein.
6. The protein of claim 1, wherein the N-terminal amino acid is a
functional derivative of a cysteine.
7. The protein of claim 1, wherein the protein is modified at both
the N-terminal amino acid and the C-terminal amino acid.
8. The protein of claims 4 or 7, wherein the protein has a
hydrophobic moiety substituted for, or appended to, at least one
amino acid intermediate to the N-terminal and C-terminal amino
acids.
9. The protein of claim 1, wherein the protein has a hydrophobic
moiety substituted for, or appended to, at least one amino acid
intermediate to the N-terminal and C-terminal amino acids.
10. The protein of claim 3, wherein the lipid moiety is a fatty
acid selected from saturated and unsaturated fatty acids having
between 2 and 24 carbon atoms.
11. The protein of claim 1, wherein the protein is a hedgehog
protein obtainable from a vertebrate source.
12. The protein of claim 11, wherein the hedgehog is obtainable
from a human or rat.
13. The protein of claim 11, wherein the vertebrate hedgehog is
selected from the group consisting of Sonic, Indian, and Desert
hedgehog.
14. The protein of claim 1, further comprising a vesicle in contact
with the hydrophobic moiety.
15. The protein of claim 14, wherein the vesicle is selected from
the group consisting of a cell membrane, a micelle, and a
liposome.
16. The protein of claim 11, wherein the hedghog protein has an
amino acid sequence according to any one of SEQ ID NOS: 1-4.
17. The protein of claim 13, wherein the hedgehog protein is
missing between 1 and about 10 amino acids from the C-terminus
thereof, when compared to a wild-type hedgehog protein.
18. The protein of claim 16, wherein the protein has at least 60%
amino acid identity to Sonic, Indian or Desert hedgehog.
19. An isolated, protein of the form: A-Cys-[Sp]-B-[Sp]-X, wherein
A is a hydrophobic moiety; Cys is a cysteine or functional
equivalent thereof; [Sp] is an optional spacer peptide sequence; B
is a protein comprising a plurality of amino acids and, optionally,
another spacer peptide sequence; and X is optionally another
hydrophobic moiety linked to an amino acid of protein B.
20. The isolated protein of claim 19, wherein the isolated protein
is a hedgehog protein.
21. The isolated protein of claim 20, wherein, if X is present,
then it is cholesterol.
22. The isolated protein of claim 19, wherein protein B is modified
at at least one other amino acid with at least one hydrophobic
moiety.
23. The isolated protein of claim 19, wherein the A-Cys linkage is
via an amino group of cysteine.
24. The isolated protein of claim 19, further comprising a vesicle
in contact therewith.
25. The isolated protein of claim 24, wherein the vesicle in
contact therewith is selected from the group consisting of a cell
membrane, micelle and liposome.
26. A vesicle to which is attached a plurality of molecules, at
least two of the plurality having the form of claim 19.
27. The vesicle of claim 26, wherein the vesicle is selected from
the group consisting of a cell membrane, liposome and micelle.
28. An isolated, protein having a C-terminal amino acid and an
N-terminal thioproline group, said group formed by reacting an
aldehyde with an N-terminal cysteine of the protein.
29. An isolated, protein having a C-terminal amino acid and an
N-terminal amide group, said group formed by reacting a fatty acid
thioester with an N-terminal cysteine of the protein.
30. An isolated, protein having a C-terminal amino acid and an
N-terminal maleimide group, said N-terminal maleimide group formed
reacting a maleimide group with the N-terminal cysteine of the
protein.
31. The isolated protein of claims 28, 29 or 30, wherein the
C-terminal amino acid of the protein is modified with an
hydrophobic moiety.
32. The isolated protein of claim 31, wherein the protein is a
hedgehog protein.
33. The isolated protein of claim 32, wherein the C-terminal
hydrophobic moiety is cholesterol.
34. A method of generating a multivalent protein complex comprising
the step of linking, in the presence of a vesicle, a hydrophobic
moiety to an N-terminal cysteine of a protein, or a functional
equivalent of the N-terminal cysteine.
35. The method of claim 34, wherein the step of linking comprises
linking a lipid moiety which is selected from saturated and
unsaturated fatty acids having between 2 and 24 carbon atoms.
36. The method of claim 34, wherein the protein is a hedgehog
protein.
37. The method of claim 36, wherein the hedgehog is selected from
the group consisting of Sonic, Indian and Desert hedgehog.
38. The method of claim 36, wherein the hedghog has an amino acid
sequence according to any one of SEQ ID NOS: 1-4.
39. The method of claim 34, wherein the step of linking comprises
linking with a vesicle selected from the group consisting of a cell
membrane, liposome and micelle.
40. A method for modifying a physico-chemical property of a
protein, comprising introducing at least one hydrophobic moiety to
an N-terminal cysteine of the proteinor to a functional equivalent
of the N-terminal cysteine.
41. The method of claim 40, further comprising contacting the
hydrophobic moiety with a vesicle.
42. The method of claim 40, wherein the hydrophobic moiety is
either a lipid moiety selected from saturated and an unsaturated
fatty acids having between 2 and 24 carbon atoms or is a
hydrophobic protein.
43. The method of claim 40, wherein the protein is a hedgehog
protein.
44. The method of claim 43, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian and Desert
hedgehog.
45. The method of claim 43, wherein the hedgehog has an amino acid
sequence according to any one of SEQ ID NOS: 1-4.
46. The method of claim 41, wherein the step of contacting
comprises contacting with a vesicle selected from the group
consisting of a cell membrane, liposome and micelle.
47. A protein complex, produced by the method of claim 34.
48. A modified protein, produced by the method of claim 40.
49. The complex of claim 47, wherein the protein is selected from
the group consisting of gelsolin; an interferon, an interleukin,
tumor necrosis factor, monocyte colony stimulating factor,
granulocyte colony stimulating factor, granulocyte macrophage
colony stimulating factor, erythropoietin, platelet derived growth
factor, growth hormone and insulin.
50. A method for modifying a protein having a biological activity
and containing an N-terminal cysteine, comprising reacting the
N-terminal cysteine with a fatty acid thioester to form an amide,
wherein such modification enhances the protein's biological
activity.
51. The method of claim 50, wherein the protein is a hedgehog
protein.
52. The method of claim 51, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian, Desert
hedgehog, and functional variants thereof.
53. A method for modifying a protein having a biological activity
and containing an N-terminal cysteine, comprising reacting the
N-terminal cysteine with a maleimide group, wherein such
modification enhances the protein's biological activity.
54. The method of claim 53, wherein the protein is a hedgehog
protein.
55. The method of claim 54, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian, Desert
hedgehog, and functional variants thereof.
56. A method for modifying a protein having a biological activity
comprising appending an hydrophobic peptide to the protein.
57. The method of claim 56, wherein the hydrophobic peptide is
appended to an amino acid of the protein selected from the group
consisting of the N-terminal amino acid, the C-terminal amino acid,
an amino acid intermediate between the N-terminal amino acid and
the C-terminal amino acid, and combinations of the foregoing.
58. The method of claim 69, wherein the protein is a hedgehog
protein.
59. The method of claim 71, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian and Desert
hedgehog.
60. A therapeutic use of the protein of any of claims 1 or 20,
comprising administering the protein to a subject.
61. A method of treating a neurological disorder in a patient
comprising administering to the patient a protein of any of claims
1 or 20.
62. The protein of claim 1, wherein the protein is an extracellular
signaling protein.
63. The method of claim 57, wherein the step of appending comprises
replacing at least the N-terminal amino acid of the protein with at
least one hydrophobic amino acid.
64. The method of claim 63, wherein the at least one hydrophobic
amino acid is a plurality of isoleucine residues.
65. The method of claim 63, further comprising chemically modifying
at least one of the isoleucine residues.
66. An isolated, protein having a C-terminal amino acid and an
N-terminal acetamide group, said group formed by reacting a
substituted acetamide with an N-terminal cysteine of the
protein.
67. An isolated, protein having a C-terminal amino acid and an
N-terminal thiomorpholine group, said group formed by reacting a
haloketone group with an N-terminal cysteine of the protein.
68. A method for modifying a protein having a biological activity
and containing an N-terminal cysteine, comprising reacting the
N-terminal cysteine with a substituted acetamide group, wherein
such modification enhances the protein's biological activity.
69. The method of claim 68, wherein the protein is a hedgehog
protein.
70. The method of claim 69, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian, Desert
hedgehog, and functional variants thereof.
71. A method for modifying a protein having a biological activity
and containing an N-terminal cysteine, comprising reacting the
N-terminal cysteine with a aloketone group, wherein such
modification enhances the protein's biological activity.
72. The method of claim 71, wherein the protein is a hedgehog
protein.
73. The method of claim 72, wherein the hedgehog protein is
selected from the group consisting of Sonic, Indian, Desert
hedgehog, and functional variants thereof.
74. A hedgehog polypeptide modified with one or more lipophilic
moieties with the proviso that, in the instance wherein the
hedgehog polypeptide is the mature N-terminal proteolytic fragment
of a hedgehog protein, the lipophilic moiety is other than a sterol
at the C-terminal residue.
75. A hedgehog polypeptide modified with one or more lipophilic
moieties at internal amino acid residues.
76. A hedgehog polypeptide modified with one or more lipophilic
aromatic hydrocarbons.
77. The hedgehog polypeptide of any of claims 74-76 which
polypeptide is provided as a purified protein preparation.
78. The hedgehog polypeptide of any of claims 74-76 which
polypeptide is provided as a pharmaceutical preparation.
79. The hedgehog polypeptide of claim 74 or 75, wherein the
lipophilic moieties are selected from the group consisting of fatty
acids, lipids, esters, alcohols, cage structures, and aromatic
hydrocarbons.
80. The hedgehog polypeptide of claim 76 or 79, wherein the
aromatic hydrocarbon is selected from the group consisting of
benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene,
chrysene, and naphthacene.
81. The hedgehog polypeptide of claim 80, wherein the aromatic
hydrocarbon is a pyrene.
82. The hedgehog polypeptide of claim 74 or 75, wherein the
lipophilic moieties are selected from the group consisting of
isoprenoids, terpenes and polyalicyclic hydrocarbons.
83. The hedgehog polypeptide of claim 82, wherein the lipophilic
moieties are selected from the group consisting of adamantanes,
buckminsterfullerenes, vitamins, polyethylene glycol, oligoethylene
glycol, (C1-C18)-alkyl phosphate diesters,
--O--CH2--CH(OH)--O--(C12-C18)- -alkyl.
84. The hedgehog polypeptide of claim 83, wherein the lipophilic
moieties are selected from the group consisting of 1- or
2-adamantylacetyl, 3-methyladamant-1-ylacetyl,
3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,
camphaneacetyl, noradamantylacetyl, norbornaneacetyl,
bicyclo[2.2.2.]-oct-5-eneacetyl,
1-methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl,
cis-5-norbornene-endo-2,3-- dicarbonyl, 5-norbornen-2-ylacetyl,
(1R)-(-)-myrtentaneacetyl, 2-norbornaneacetyl,
anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-car- bonyl,
decanoyl, dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl and
dodecynoyl.
85. The hedgehog polypeptide of any of claims 74-76, wherein the
lipophilic moiety or moieties potentiate the biological activity of
the polypeptide relative to the modified hedgehog polypeptide.
86. A method for altering the growth state of a cell responsive to
hedgehog signaling, comprising contacting the cell with a
lipophilic-modified hedgehog polypeptide of any of claims 74-76.
Description
BACKGROUND OF THE INVENTION
[0001] It is known that certain proteins exhibit greater biological
activity when attached to other moieties, either by formation of
multimeric complexes, where the proteins have an opportunity to act
in concert, or through other alterations in the protein's
physico-chemical properties, such as the protein's absorption,
biodistribution and half life. Thus, one current area of research
in biotechnology involves the development of methods to modify the
physico-chemical properties of proteins so that they can be
administered in smaller amounts, with fewer side effects, by new
routes, and with less expense.
[0002] For example, the binding affinity of any single protein
(such as a ligand for its cognate receptor) may be low. However,
cells normally express hundreds to thousands of copies of a
particular surface receptor, and many receptor-ligand interactions
take place simultaneously. When many surface molecules become
involved in binding, the total effective affinity is greater than
the sum of the binding affinities of the individual molecules. By
contrast, when ligand proteins are removed from the cell surface
and purified, or isolated by recombinant DNA techniques for use,
e.g., as therapeutics, they act as monomers and lose the advantage
of acting in concert with many other copies of the same protein
associated closely on the surface of a cell. Thus isolated, the low
affinity of a protein for its receptor may become a serious
drawback to its effectiveness as a therapeutic to block a
particular binding pathway, since it must compete against the high
avidity cell-cell interactions. Effective treatment might require
constant administration and/or high doses. Such drawbacks might be
avoided, however, if a means could be found to provide multimeric
forms of an isolated protein.
[0003] Similarly, it would be useful to modify other
physico-chemical properties of biologically active proteins so
that, for instance, a protein is induced to associate with a
membrane thus localizing it at the site of administration and
enhancing its ability to bind to, or otherwise interact with, a
particular target. Such changes may also affect the
pharmaco-distribution of the protein.
[0004] Several methods of generating coupled proteins have been
developed. Many of these methods are not highly specific, i.e.,
they do not direct the point of coupling to any particular site on
the protein. As a result, conventional coupling agents may attack
functional sites or sterically block active sites, rendering the
coupled proteins inactive. Furthermore, the coupled products may be
oriented so that the active sites cannot act synergistically,
thereby rendering the products no more effective than the monomeric
protein alone.
[0005] As an additional motivation to find new methods for protein
modification, proteins with an N-terminal cysteine residue are
susceptible to oxidation or other chemical modifications that may
compromise activity or half-life. Additionally, certain buffers
commonly used in protein purification have components or impurities
that can modify the N-terminal cysteine. Even when these buffers
are avoided, the N-terminal cysteine is modified over time, perhaps
due to chemicals in the storage vials or in the air. Consequently,
formulation buffers must include a protective agent, such as
dithiothreitol, to prevent cysteine modification and/or oxidation.
However, protective agents have significant biological activity of
their own and they may therefore complicate experiments and
adversely affect the therapeutic utility of a formulation.
[0006] Accordingly, there is a need in the art to develop more
specific means to obtain derivatized products or multimeric forms
thereof so as to alter the properties of the protein in order to
affect its stability, potency, pharmacokinetics, and
pharmacodynamics.
SUMMARY OF THE INVENTION
[0007] In one aspect of the invention, we have solved the problem
of finding a way to conveniently make modified forms of
biologically active proteins. Methods of the invention can be used
to derive multimeric forms of the proteins and/or can be used to
change their physico-chemical properties. We have found that
modifying a protein (i.e, adding or appending a hydrophobic moiety
to an existing amino acid or substituting a hydrophobic moiety for
an amino acid) so as to introduce the hydrophobic moiety onto a
protein can increase the protein's biological activity and/or its
stability. For example, an N-terminal cysteine can be used as a
convenient "target" to attach a hydrophobic moiety (e.g., a lipid)
and thereby modify biologically active proteins.
[0008] Alternatively, a hydrophobic moiety can be attached to a
C-terminal residue of a biologically active protein, such as
hedgehog protein, to modify the protein's activity. A hydrophobic
moiety can also be appended to an internal amino acid residue to
enhance the protein's activity, provided the modification does not
affect the activity of the protein, e.g., the proteins ability to
bind to a receptor or co-receptor, or affect the protein's
3-dimensional structure. Preferably, the hydrophobic moiety is
appended to an internal amino acid residue that is on the surface
of the protein when the protein is in its native form. The
hydrophobic modification of the invention provides a generically
useful method of creating proteins with altered physico-chemical
properties as compared to non-modified forms.
[0009] This invention originated from the discovery that when we
expressed full-length Sonic hedgehog protein in insect and in
mammalian cells, the mature form of the protein (residues 1-174 in
the mature sequence), in addition to having cholesterol at the
C-terminus, is also derivatized at its N-terminal end with a fatty
acid. Significantly, this form of hedgehog exhibited about a
30-fold increase in potency as compared to soluble, unmodified
hedgehog in an in vitro assay.
[0010] One aspect of the invention is therefore an isolated,
protein comprising an N-terminal amino acid and a C-terminal amino
acid, wherein the protein is selected from the group consisting of
a protein with an N-terminal cysteine that is appended with at
least one hydrophobic moiety; a protein with an N-terminal amino
acid that is not a cysteine appended with a hydrophobic moiety; and
a protein with a hydrophobic moiety substituted for the N-terminal
amino acid. The hydrophobic moiety can be a hydrophobic peptide or
any lipid or any other chemical moiety that is hydrophobic.
[0011] The protein may be modified at its N-terminal amino acid and
preferably the N-terminal amino acid is a cysteine or a functional
derivative thereof. The protein may be modifed at its C-terminal
amino acid or at both the N-terminal amino acid and the C-terminal
amino acid, or at at least one amino acid internal to (i.e.,
intermediate between) the N-terminal and C-terminal amino acids, or
various combinations of these configurations. The protein can be an
extracellular signaling protein and in preferred embodiments, the
protein is a hedgehog protein obtainable from a vertebrate source,
most preferably obtainable from a human and includes Sonic, Indian,
and Desert hedgehog.
[0012] Another embodiment is an isolated, protein of the form:
A-Cys-[Sp]-B-X, wherein A is a hydrophobic moiety;
[0013] Cys is a cysteine or functional equivalent thereof;
[0014] [Sp] is an optional spacer peptide sequence;
[0015] B is a protein comprising a plurality of amino acids,
including at least one optional spacer peptide sequence; and
[0016] X is optionally another hydrophobic moiety linked to the
protein.
[0017] The isolated protein can be an extracellular signaling
protein, preferably a hedgehog protein. This protein can be
modified at at least one other amino acid with at least one
hydrophobic moiety. In other embodiments, the protein is in contact
with a vesicle in selected from the group consisting of a cell
membrane, micelle and liposome.
[0018] Another aspect of the invention is an isolated, protein
having a C-terminal amino acid and an N-terminal thiaproline group,
the thiaproline group formed by reacting an aldehyde with an
N-terminal cysteine of the protein. A further aspect of the
invention is isolated, protein having a C-terminal amino acid and
an N-terminal amide group, the amide group formed by reacting a
fatty acid thioester with an N-terminal cysteine of the protein.
Yet another aspect of the invention is an isolated, protein having
a C-terminal amino acid and an N-terminal maleimide group, the
N-terminal maleimide group formed by reacting a maleimide group
with the N-terminal cysteine of the protein. Yet another aspect of
the invention is an isolated, protein having a C-terminal amino
acid and an N-terminal acetamide group. A further aspect of the
invention is an isolated, protein having a C-terminal amino acid
and an N-terminal thiomorpholine group.
[0019] In these embodiments, the C-terminal amino acid of the
protein can be modified with an hydrophobic moiety. The isolated
protein can be an extracellular signaling protein, most preferably
a hedgehog protein.
[0020] Methods of the invention include a method of generating a
multivalent protein complex comprising the step of linking, in the
presence of a vesicle, a hydrophobic moiety to an N-terminal
cysteine of a protein, or a functional equivalent of the N-terminal
cysteine. The linking step may include linking a lipid moiety which
is selected from saturated and unsaturated fatty acids having
between 2 and 24 carbon atoms. The protein can be an extracellular
signaling protein, preferably a hedgehog protein selected from the
group consisting of Sonic, Indian and Desert hedgehog.
[0021] Yet another method of the invention is a method for
modifying a physico-chemical property of a protein, comprising
introducing at least one hydrophobic moiety to an N-terminal
cysteine of the protein or to a functional equivalent of the
N-terminal cysteine. The hydrophobic moiety can be a lipid moiety
selected from saturated and unsaturated fatty acids having between
2 and 24 carbon atoms. It can also be a hydrophobic protein The
protein modified using this method can be an extracellular
signaling protein, preferably a hedgehog protein selected from the
group consisting of Sonic, Indian and Desert hedgehog. A protein
complex, produced by these methods are also encompassed by the
present invention.
[0022] Other extracellular signaling proteins besides hedgehog
include gelsolin; an interferon, an interleukin, tumor necrosis
factor, monocyte colony stimulating factor, granulocyte colony
stimulating factor, granulocyte macrophage colony stimulating
factor, erythropoietin, platelet derived growth factor, growth
hormone and insulin.
[0023] Another method is a method for modifying a protein (such as
an extracellular signaling protein) that has an N-terminal
cysteine. This method comprises reacting the N-terminal cysteine
with a fatty acid thioester to form an amide, wherein such
modification enhances the protein's biological activity.
[0024] Yet another method is a method for modifying a protein (such
as an extracellular signaling protein) having an N-terminal
cysteine, which comprising reacting the N-terminal cysteine with a
maleimide group, wherein such modification enhances the protein's
biological activity. Other embodiments of this method involve
reacting the N-terminal cysteine with either an aldehyde group, an
acetamide group or a thiomorpholine group.
[0025] A further method is a method for modifying protein (such as
an extracellular signaling protein) comprising appending an
hydrophobic peptide to the protein. The hydrophobic moiety can be
appended to an amino acid of the protein selected from the group
consisting of the N-terminal amino acid, the C-terminal amino acid,
an amino acid intermediate between the N-terminal amino acid and
the C-terminal amino acid, and combinations of the foregoing. In
one embodiment, the present invention provides hedgehog
polypeptides which are modified with lipophilic moieties. In
certain embodiments, the hedgehog proteins of the present invention
are modified by a lipophilic moiety or moieties at one or more
intenal sites of the mature, processed extracellular domain, and
may or may not be also derivatized with lipophilic moieties at the
N or C-terminal residues of the mature polypeptide. In other
embodiments, the polypeptide is modified at the C-terminal residue
with a hydrophobic moiety other than a sterol. In still other
embodiments, the polypeptide is modified at the N-terminal residue
with a cyclic (preferably polycyclic) lipophilic group. Various
combinations of the above are also contemplated. A therapeutic
method of the invention is a method for treating a neurological
disorder in a patient comprising administering to the patient a
hydrophobically-modified protein of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1. Characterization of a palmitoylated form of Shh. A
tethered form of human Shh was immunoaffinity purified from High
Five.TM. insect cells and analyzed by SDS-PAGE. The protein was
stained with Coomassie blue (lane a, Life Technologies, Inc.
prestained high molecular weight markers; lane b, soluble Shh (0.6
.mu.g); lane c, tethered Shh (0.6 .mu.g); lane d, mixture of
soluble plus tethered Shh (0.6 .mu.g each)). The ability of Shh and
Ihh (see lane h) to be modified with palmitic acid was assayed
using a cell-free system described in Example 2. Soluble forms of
hedgehog protein (3 .mu.g/sample) were incubated for 1 h with rat
liver microsomes, ATP, CoenzymeA, and .sup.3H-palmitic acid, and
then analyzed for palmitoylation by SDS-PAGE. The samples shown in
lanes e-i were visualized by fluorography (lane e, Shh; lane f, des
1-10 Shh; lane g, Cys-1 to Ser Shh; lane h, Ihh; lane i, His-tagged
Shh) and in lanes j-k by Coomassie staining (lane j, Shh; lane k
des 1-10 Shh).
[0027] FIG. 2. Analysis of purified Shh by ESI-MS. Soluble human
Shh (A) and tethered human Shh (B) were analyzed by ESI-MS on a
Micromass Quattro II triple quadrupole mass spectrometer, equipped
with an electrospray ion source. All electrospray mass spectral
data were acquired and stored in profile mode and were processed
using the Micromass MassLynx data system. Molecular mass spectra
are shown (mass assignments were generated by the data system).
[0028] FIG. 3. Analysis of tethered Shh by reverse phase HPLC.
Soluble human Shh (A), tethered human Shh from High Five.TM. insect
cells (B), tethered human Shh from EBNA-293 cells (C), and
cell-associated rat Shh (D) were subjected to reverse phase HPLC on
a narrow bore Vydac C.sub.4 column (2.1 mm internal
diameter.times.250 mm). The column was developed with a 30 min,
0-80% acetonitrile gradient in 0.1% trifluoroacetic acid at 0.25
mL/min and the effluent monitored using a photodiode array detector
from 200-300 nm (data shown at 214 nm). Peak fractions were
collected and characterized further by SDS-PAGE and MS (data
summarized in Tables 3, 4, and 5).
[0029] FIG. 4. Characterization of Shh by LC-MS. Tethered human Shh
(A) and soluble human Shh (B) were alkylated with 4-vinylpyridine
(1 .mu.L/100 .mu.L sample in 6 M guanidine HCl, 1 mM EDTA, 100 mM
Tris HCl pH 8.0), ethanol precipitated, and digested with
endoproteinase Lys-C in 50 mM Tris HCl pH 7.0, 2 M urea at an
enzyme:protein ratio of 1:5 as described previously (27). The
digests were analyzed by reverse phase HPLC in line with an
electrospray Micromass Quattro II triple quadrupole mass
spectrometer. Scans were acquired throughout the run and processed
using the Micromass MassLynx data system (total ion chromatograms
from the runs are shown). Asterisks indicate the positions of the
N-terminal peptide which were verified either by MALDI PSD or
N-terminal Edman sequencing.
[0030] FIG. 5. Sequencing of the N-terminal Shh peptide by MALDI
PSD measurement. The N-terminal endoproteinase Lys-C peptide from
tethered human Shh was subjected to MALDI PSD measurement on a
Voyager-DE.TM. STR time of flight mass spectrometer. The predicted
fragmentation pattern and nomenclature for the detected fragment
ions are shown at the top of the panel (PA, palmitoyl acid; 4vp,
4-pyridylethyl group). The remainder of the Figure shows the
molecular mass spectrum produced by the run. Relevant ions are
denoted using the nomenclature defined in the schematic. Calculated
masses (Da) for b.sub.1-b.sub.8 are 447.3, 504.3, 601.4, 658.4,
814.5, 871.5, 1018.6, and 1075.6, respectively. For
y.sub.1-y.sub.8, the masses (Da) are 147.1, 204.1, 351.2, 408.2,
564.3, 621.3, 718.4, and 775.4, respectively. The calculated mass
for z.sub.8 is 758.4 Da. The observed mass for b.sub.8 contains an
additional 18 Da due to an added water.
[0031] FIG. 6. Increased activity of tethered Shh in the C3H10T1/2
assay. The relative potencies of soluble and tethered human Shh
alone (A) or in the presence of the anti-hedgehog neutralizing Mab
5E1 (B) were assessed on C3H10T1/2 cells measuring alkaline
phosphatase induction. The numbers presented reflect the averages
of duplicate determinations. (A) Serial 2-fold dilutions of soluble
(6) and tethered (8) Shh were incubated with the cells for 5 days
and the resulting levels of alkaline phosphatase activity measured
at 405 nm using the alkaline phosphatase chromogenic substrate
p-nitrophenyl phosphate. (B) Serial dilutions of Mab 5E1 were
incubated with soluble Shh (5 .mu.g/mL: black bars) or tethered Shh
(0.25 .mu.g/mL: gray bars) or vehicle control without Shh added
(white bar) for 30 min and then subjected to analysis in the
C3HT101/2 assay.
[0032] FIG. 7. Analysis of Shh in a receptor binding assay. The
relative potency of soluble (6) and tethered (8) Shh for binding to
patched was assessed on patched-transfected EBNA-293 cells by FACS
analysis. Serial dilutions of the test samples were incubated with
the EBNA-293 cells, washed, and then the percent binding measured
by the ability of the samples to compete with Shh-Ig for binding to
the cells. Bound Shh-Ig was quantified by mean fluorescence using a
FITC-labeled anti-Ig antibody probe as the readout. The data were
fitted to a hyperbolic curve by non-linear regression.
[0033] FIG. 8. Alignment of N-terminal fragment of human hedgehog
proteins. The 20 kDa human hedgehog proteins (Sonic "Shh", Desert
"Dhh" and Indian "Ihh") are aligned with respect to their
N-terminal cysteine (Cys-1 in the mature sequence). This cysteine
is normally Cys-24 in the full-length Shh precursor protein due to
the presence of the natural signal sequence that is removed during
secretion. The actual position of the cysteine may vary slightly
due to species differences.
[0034] FIG. 9. Consensus Sequence of the N-terminal fragment of
human hedgehog proteins.
[0035] FIG. 10. Effect of lipid chain length on activity of human
Sonic hedgehog. A series of fatty acid-modified hedgehog proteins
was synthesized according to the present invention and the effect
of the fatty acid chain length on hedgehog activity was tested
using the C3H10T1/2 alkaline phosphatase induction assay described
herein. The results are plotted as a bar graph.
[0036] FIG. 11. C3H10T1/2 assay of palmitoylated, myristyolated,
lauroylated, decanoylated, and octanoylated human Sonic hedgehog.
Palmitoylated, lauroylated, decanoylated, and octanoylated human
Sonic hedgehog formulated in 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM
NaCl, 1% octylglucoside, 0.5 mM DTT, and myristoylated human Sonic
hedgehog, formulated in 1-50 mM NaCl, 0.5 mM DTT, were assayed on
C3H10T1/2 cells measuring alkaline phosphatase induction. The
numbers represent the mean of duplicate determinations. Serial
3-fold dilutions of palmitoylated (.largecircle.), myristoylated
(.circle-solid.), lauroylated (.quadrature.), decanoylated
(.box-solid.), octanoylated (.DELTA.), and unmodified
(.tangle-solidup. and x) human Sonic hedgehog were incubated with
the cells for 5 days and the resulting levels of alkaline
phosphatase measured at 405 nm using the chromogenic substrate
p-nitrophenyl phosphate. The palmitoylated, myristoylated,
lauroylated, and decanoylated proteins were assayed in one
experiment with the unmodified protein shown as (.tangle-solidup.),
while the octanoylated protein was assayed in another experiment
with the unmodified protein shown as (x). The arrow on the y-axis
denotes the background level of alkaline phosphatase in the absence
of added hedgehog protein.
[0037] FIG. 12. Generic structures of various
hydrophobically-modified forms of hedgehog. (A) Fatty amide
derivative where R=a hydrocarbon chain of a fatty acid; (B)
thiazolidine derivative where R=a hydrocarbon; (C) amino acid
substitution where R=a hydrophobic amino acid side chain; (D)
maleimide derivative where R=a hydrocarbon; (E) SH=free thiol on
N-terminal cysteine of wild type hedgehog; (F) an iodoacetamide
derivative where R.sub.1=a hydrocarbon and R.sub.2=either H or a
hydrocarbon; and (G) thiomorpholinyl derivative where R=a
hydrocarbon. For all structures, HH=hedgehog.
[0038] FIG. 13. Relative potency of various
hydrophobically-modified forms of hedgehog in the C3H10T1/2 assay.
The EC.sub.50 (2 .mu.g/ml) of unmodified wild type human Sonic
hedgehog is designated as 1 x. The potency of the other proteins is
expressed as the ratio of the EC.sub.50 of wild type protein
divided by the EC.sub.50 of the modified protein. Modifications are
at the N-terminus of the protein unless designated otherwise.
[0039] FIG. 14. Relative potency of the unmodified, myristoylated,
and C1 II mutant of human Sonic hedgehog in a malonate-induced rat
striatal lesion assay. The figure shows the reduction in
malonate-induced lesion volume which results from the administation
of either unmodified, myristoylated, or the C1 II mutant of human
Sonic hedegehog to the rat striatum.
[0040] FIG. 15. illustrates the specific activities of maleimide
modified and unmodified hedgehog polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
[0041] This invention is based, in part, on the discovery that
human Sonic hedgehog, expressed as a full-length construct in
either insect or in mammalian cells, has a hydrophobic palmitoyl
group appended to the .alpha.-amine of the N-terminal cysteine.
This is the first example, of which the inventors are aware, of an
extracellular signaling protein being modified in such a manner,
and, in contrast to thiol-linked palmitic acid modifications whose
attachment is readily reversible, this novel N-linked palmitoyl
moiety is likely to be very stable by analogy with myristic acid
modification.
[0042] As a direct consequence of this initial discovery, the
inventors have found that increasing the hydrophobic nature of a
signaling protein can increase the protein's biological activity.
In particular, the inventors have found that appending a
hydrophobic moiety to a signaling protein, such as a hedgehog
protein, can enhance the protein's activity. The inventors have
found that the N-terminal cysteine of biologically active proteins
not only provides a convenient site for appending a hydrophobic
moeity, and thereby modifying the physico-chemical properties of
the protein, but modifications to the N-terminal cysteine can also
increase the protein's stability. Additionally, addition of a
hydrophobic moiety to an internal amino acid residue on the surface
of the protein structure enhances the protein's activity. We use as
an example, our discovery of hydrophobic (e.g., lipids and
hydrophobic amino acid) modifications of hedgehog protein.
[0043] One aspect of the present application is directed to the
discovery that, in addition to those effects seen by
cholesterol-addition to the C-terminus of extracellular fragments
of the protein, at least certain of the biological activities of
the hedgehog gene products are unexpectedly potentiated by
derivativation of the protein with lipophilic moieties at other
sites on the protein and/or by moieties other than cholesterol.
Certain aspects of the invention are directed to preparations of
hedgehog polypeptides which are modified at sites other than
N-terminal or C-terminal residues of the natural processed form of
the protein, and/or which are modified at such terminal residues
with lipophilic moieties other than a sterol at the C-terminus or
fatty acid at the N-terminus.
[0044] As described in PCT publications WO 95/18856 and WO 96/17924
(all of which are expressly incorporated by reference herein),
hedgehog polypeptides in general are useful in the in vitro and in
vivo repairing and/or regulating the functional performance of a
wide range of cells, tissues and organs, and have therapeutic uses
ragning from neuroprotection, neuroregeneration, enhancement of
neural function, regulation of bone and cartilage formation and
repair, regulation of spermatogenesis, regulation of lung, liver
and other organs arising from the primative gut, regulation of
hematopoietic function, etc. Accordingly, the methods and
compositions of the present invention include the use of the
derivatized hedgehog polypeptides for all such uses as hedgehog
proteins have been implicated. Moreover, the subject methods can be
performed on cells which are provided in culture (in vitro), or on
cells in a whole animal (in vivo).
[0045] In one aspect, the present invention provides pharmaceutical
preparations comprising, as an active ingredient, a hedgehog
polypeptide being derivatized by one or more lipophilic moieties
such as described herein.
[0046] The subject hedgehog treatments are effective on both human
and animal subjects. Animal subjects to which the invention is
applicable extend to both domestic animals and livestock, raised
either as pets or for commercial purposes. Examples are dogs, cats,
cattle, horses, sheep, hogs and goats.
[0047] The hedgehog proteins are a family of extracellular
signaling proteins that regulate various aspects of embryonic
development both in vertebrates and in invertebrates (for reviews
see 1,2). The most well-characterized hedgehog protein is Sonic
hedgehog (Shh), involved in anterior-posterior patterning,
formation of an apical ectodermal ridge, hindgut mesoderm, spinal
column, distal limb, rib development, and lung development, and in
inducing ventral cell types in the spinal cord, hindbrain and
forebrain (3-8). While the mechanism of action of hedgehog proteins
is not understood fully, the most recent biochemical and genetic
data suggest that the receptor for Shh is the product of the tumor
suppressor gene, patched (9,10) and that other proteins; smoothened
(10,11), Cubitus interruptus (12,13), and fused (14) are involved
in the hedgehog signaling pathway.
[0048] Human Shh is synthesized as a 45 kDa precursor protein that
is cleaved autocatalytically to yield: (I) a 20 kDa N-terminal
fragment that is responsible for all known hedgehog signaling
activity (SEQ ID NOS. 1-4); and (II) a 25 kDa C-terminal fragment
that contains the autoprocessing activity (15-17). The N-terminal
fragment consists of amino acid residues 24-197 of the full-length
precursor sequence.
[0049] The N-terminal fragment remains membrane-associated through
the addition of a cholesterol at its C-terminus (18,19). This
cholesterol is critical for restricting the tissue localization of
the hedgehog signal. The addition of the cholesterol is catalyzed
by the C-terminal domain during the processing step.
[0050] All references cited in the detailed description are, unless
otherwise stipulated, incorporated herein by reference.
[0051] I. Definitions
[0052] The invention will now be described with reference to the
following detailed description of which the following definitions
are included:
[0053] "amino acid"--a monomeric unit of a peptide, polypeptide, or
protein. There are twenty amino acids found in naturally occurring
peptides, polypeptides and proteins, all of which are L-isomers.
The term also includes analogs of the amino acids and D-isomers of
the protein amino acids and their analogs.
[0054] "protein"--any polymer consisting essentially of any of the
20 amino acids. Although "polypeptide" is often used in reference
to relatively large polypeptides, and "peptide" is often used in
reference to small polypeptides, usage of these terms in the art
overlaps and is varied. The term "protein" as used herein refers to
peptides, proteins and polypeptides, unless otherwise noted.
[0055] "N-terminal end"--refers to the first amino acid (amino acid
number 1) of the mature form of a protein.
[0056] "N-terminal cysteine"--refers to the amino acid residue
(number 1) as shown in SEQ ID NOS. 1-4. It also refers to any
cysteine at position 1 of any other protein, or functional
equivalents of this cysteine (See Section IV).
[0057] "spacer" sequence refers to a short sequence that can be as
small as a single amino acid that may be inserted between an amino
acid to be hydrophobically modified (such as, for example, the
N-terminal cysteine or functional equivalent) and the remainder of
the protein. A spacer is designed to provide separation between
the-hydrophobic modification (e.g., the modified N-terminal
cysteine) and the rest of the protein so as to prevent the
modification from interfering with protein function and/or make it
easier for the modification (e.g., the N-terminal cysteine) to link
with a lipid, vesicle, or other hydrophobic moiety. Thus, if a
protein is modified at its N-terminal cysteine and at an amino acid
at another site, there may be two, or more, spacer sequences.
[0058] "tethered" protein--refers to a hydrophobically-modified
protein according to the invention.
[0059] "multivalent protein complex"--refers to a plurality of
proteins (i.e., one or more). A lipid or other hydrophobic moiety
is attached to at least one of the plurality of proteins. The lipid
or other hydrophobic moiety may optionally be in contact with a
vesicle. If a protein lacks a lipid or other hydrophobic moiety,
then that protein may be cross-linked or bind to a protein that
does have a lipid or other hydrophobic moiety. Each protein may be
the same or different and each lipid or other hydrophobic moiety
may be the same or different.
[0060] "vesicle"--refers to any aggregate of lipophilic molecules.
The vesicle may be obtained from a biologic source (e.g., a lipid
bilayer such as a cell membrane or a cholic acid-derived detergent
preparation) or from a non-biologic source (e.g., a non-biologic
detergent vesicle as described in Section VI). The shape, type, and
configuration of the vesicle is not intended to limit the scope of
this invention.
[0061] "functional equivalent" of an amino acid residue (e.g., an
N-terminal cysteine)--is (i) an amino acid having similar reactive
properties as the amino acid residue that was replaced by the
functional equivalent; (ii) an amino acid of a ligand of a
polypeptide of the invention, the amino acid having similar
hydrophobic (e.g., lipid) moiety binding properties as the amino
acid residue that was replaced by the functional equivalent; (iii)
a non-amino acid molecule having similar hydrophobic (e.g., lipid)
moiety binding properties as the amino acid residue that was
replaced by the functional equivalent.
[0062] "genetic fusion"--refers to a co-linear, covalent linkage of
two or more proteins or fragments thereof via their individual
peptide backbones, through genetic expression of a polynucleotide
molecule encoding those proteins.
[0063] A "chimeric protein" or "fusion protein" is a fusion of a
first amino acid sequence encoding a hedgehog polypeptide with a
second amino acid sequence defining a domain foreign to and not
substantially homologous with any domain of hh protein. A chimeric
protein may present a foreign domain which is found (albeit in a
different protein) in an organism which also expresses the first
protein, or it may be an "interspecies", "intergenic", etc. fusion
of protein structures expressed by different kinds of organisms. In
general, a fusion protein can be represented by the general formula
(X).sub.n-(hh).sub.m-(Y).sub.n, wherein hh represents all or a
portion of the hedgehog protein, X and Y each independently
represent an amino acid sequences which are not naturally found as
a polypeptide chain contiguous with the hedgehog sequence, m is an
integer greater than or equal to 1, and each occurrence of n is,
independently, 0 or an integer greater than or equal to 1 (n and m
are preferably no greater than 5 or 10).
[0064] "mutant"--any change in the genetic material of an organism,
in particular any change (i.e., deletion, substitution, addition,
or alteration) in a wild type polynucleotide sequence or any change
in a wild type protein.
[0065] "wild type"--the naturally-occurring polynucleotide sequence
of an exon of a protein, or a portion thereof, or protein sequence,
or portion thereof, respectively, as it normally exists in
vivo.
[0066] "standard hybridization conditions"--salt and temperature
conditions substantially equivalent to 0.5.times.SSC to about
5.times.SSC and 65.degree. C. for both hybridization and wash. The
term "standard hybridization conditions" as used herein is an
operational definition and encompasses a range of hybridization
conditions. See also Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989).
[0067] "expression control sequence"--a sequence of polynucleotides
that controls and regulates expression of genes when operatively
linked to those genes.
[0068] "operatively linked"--a polynucleotide sequence (DNA, RNA)
is operatively linked to an expression control sequence when the
expression control sequence controls and regulates the
transcription and translation of that polynucleotide sequence. The
term "operatively linked" includes having an appropriate start
signal (e.g., ATG) in front of the polynucleotide sequence to be
expressed, and maintaining the correct reading frame to permit
expression of the polynucleotide sequence under the control of the
expression control sequence, and production of the desired
polypeptide encoded by the polynucleotide sequence.
[0069] "expression vector"--a polynucleotide, such as a DNA plasmid
or phage (among other common examples) which allows expression of
at least one gene when the expression vector is introduced into a
host cell. The vector may, or may not, be able to replicate in a
cell.
[0070] "Isolated" (used interchangeably with "substantially
pure")--when applied to nucleic acid i.e., polynucleotide sequences
that encode polypeptides, means an RNA or DNA polynucleotide,
portion of genomic polynucleotide, cDNA or synthetic polynucleotide
which, by virtue of its origin or manipulation: (i) is not
associated with all of a polynucleotide with which it is associated
in nature (e.g., is present in a host cell as an expression vector,
or a portion thereof); or (ii) is linked to a nucleic acid or other
chemical moiety other than that to which it is linked in nature; or
(iii) does not occur in nature. By "isolated" it is further meant a
polynucleotide sequence that is: (i) amplified in vitro by, for
example, polymerase chain reaction (PCR); (ii) synthesized
chemically; (iii) produced recombinantly by cloning; or (iv)
purified, as by cleavage and gel separation.
[0071] Thus, "substantially pure nucleic acid" is a nucleic acid
which is not immediately contiguous with one or both of the coding
sequences with which it is normally contiguous in the naturally
occurring genome of the organism from which the nucleic acid is
derived. Substantially pure DNA also includes a recombinant DNA
which is part of a hybrid gene encoding additional hedgehog
sequences.
[0072] "Isolated" (used interchangeably with "substantially
pure")--when applied to polypeptides means a polypeptide or a
portion thereof which, by virtue of its origin or manipulation: (i)
is present in a host cell as the expression product of a portion of
an expression vector; or (ii) is linked to a protein or other
chemical moiety other than that to which it is linked in nature; or
(iii) does not occur in nature, for example, a protein that is
chemically manipulated by appending, or adding at least one
hydrophobic moiety to the protein so that the protein is in a form
not found in nature. By "isolated" it is further meant a protein
that is: (i) synthesized chemically; or (ii) expressed in a host
cell and purified away from associated and contaminating proteins.
The term generally means a polypeptide that has been separated from
other proteins and nucleic acids with which it naturally occurs.
Preferably, the polypeptide is also separated from substances such
as antibodies or gel matrices (polyacrylamide) which are used to
purify it.
[0073] "Heterologous promoter"--as used herein is a promoter which
is not naturally associated with a gene or a purified nucleic
acid.
[0074] "Homologous"--as used herein is synonymous with the term
"identity" and refers to the sequence similarity between two
polypeptides, molecules, or between two nucleic acids. When a
position in both of the two compared sequences is occupied by the
same base or amino acid monomer subunit (for instance, if a
position in each of the two DNA molecules is occupied by adenine,
or a position in each of two polypeptides is occupied by a lysine),
then the respective molecules are homologous at that position. The
percentage homology between two sequences is a function of the
number of matching or homologous positions shared by the two
sequences divided by the number of positions compared.times.100.
For instance, if 6 of 10 of the positions in two sequences are
matched or are homologous, then the two sequences are 60%
homologous. By way of example, the DNA sequences CTGACT and CAGGTT
share 50% homology (3 of the 6 total positions are matched).
Generally, a comparison is made when two sequences are aligned to
give maximum homology. Such alignment can be provided using, for
instance, the method of Needleman et al., J. Mol Biol. 48: 443-453
(1970), implemented conveniently by computer programs such as the
Align program (DNAstar, Inc.). Homologous sequences share identical
or similar amino acid residues, where similar residues are
conservative substitutions for, or "allowed point mutations" of,
corresponding amino acid residues in an aligned reference sequence.
In this regard, a "conservative substitution" of a residue in a
reference sequence are those substitutions that are physically or
functionally similar to the corresponding reference residues, e.g.,
that have a similar size, shape, electric charge, chemical
properties, including the ability to form covalent or hydrogen
bonds, or the like. Particularly preferred conservative
substitutions are those fulfilling the criteria defined for an
"accepted point mutation" in Dayhoff et al., 5: Atlas of Protein
Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat.
Biomed. Res. Foundation, Washington, D.C. (1978).
[0075] A "hedgehog protein" or "hedgehog polypeptide", as the terms
are used interchangeably, of the invention is defined in terms of
having at least a portion that consists of the consensus amino acid
sequence of SEQ ID NO: 4. The term also means a hedgehog
polypeptide, or a functional variant of a hedgehog polypeptide, or
homolog of a hedgehog polypeptide, or functional variant, which has
biological activity. In particular, the terms encompasses
preparations of hedgehog proteins and peptidyl fragments thereof,
both agonist and antagonist forms as the specific context will make
clear. As used herein the term "bioactive fragment of a hedgehog
protein" refers to a fragment of a full-length hedgehog
polypeptide, wherein the fragment specifically agonizes or
antagonizes inductive events mediated by wild-type hedgehog
proteins. The hedgehog biactive fragment preferably is a soluble
extracellular portion of a hedgehog protein, where solubility is
with reference to physiologically compatible solutions. Exemplary
bioactive fragments are described in PCT publications WO 95/18856
and WO 96/17924. In preferred embodiments, the hedgehog
polypeptides of the present invention bind to the patched
protein.
[0076] The term "corresponds to", when referring to a particular
polypeptide or nucleic acid sequence is meant to indicate that the
sequence of interest is identical or homologous to the reference
sequence to which it is said to correspond.
[0077] The terms "peptide(s)", "protein(s)" and "polypeptide(s)"
are used interchangeably herein. The terms "polynucleotide
sequence" and "nucleotide sequence" are also used interchangeably
herein. The terms "Hedgehog fragment" and "Hedgehog N-terminal
fragment" are used interchangeably with "Hedgehog".
[0078] A hedgehog molecule has "biological activity" if it has at
least one of the following properties: (i) the molecule meets the
hedgehog consensus criteria as defined herein (SEQ ID NO: 4) and
has the ability to bind to its receptor, patched or it encodes,
upon expression, a polypeptide that has this characteristic; (ii)
the molecule meets the hedgehog consensus criteria as defined
herein or it encodes, upon expression, a polypeptide that has this
characteristic; and (iii) it may induce alkaline phosphatase
activity in C3H10T1/2 cells. Generally, any protein has "biological
activity" if the protein has in vitro effects, properties, or
characteristics that persons having ordinary skill in the art would
recognize as being representative of, commensurate with, or
reasonably predictive of, the protein's in vivo effects.
[0079] The term "hydrophobic" refers to the tendency of chemical
moieties with nonpolar atoms to interact with each other rather
than water or other polar atoms. Materials that are "hydrophobic"
are, for the most part, insoluble in water. Natural products with
hydrophobic properties include lipids, fatty acids, phospholipids,
sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes,
prostaglandins, thromboxanes, leukotrienes, isoprenoids, retenoids,
biotin, and hydrophobic amino acids such as tryptophan,
phenylalanine, isoleucine, leucine, valine, methionine, alanine,
proline, and tyrosine. A chemical moiety is also hydrophobic or has
hydrophobic properties if its physical properties are determined by
the presence of nonpolar atoms. The term includes lipophilic
groups.
[0080] The term "lipophilic group", in the context of being
attached to a polypeptide, refers to a group having high
hydrocarbon content thereby giving the group high affinity to lipid
phases. A lipophilic group can be, for example, a relatively long
chain alkyl or cycloalkyl (preferably n-alkyl) group having
approximately 7 to 30 carbons. The alkyl group may terminate with a
hydroxy or primary amine "tail". To further illustrate, lipophilic
molecules include naturally-occurring and synthetic aromatic and
non-aromatic moieties such as fatty acids, esters and alcohols,
other lipid molecules, cage structures such as adamantane and
buckminsterfullerenes, and aromatic hydrocarbons such as benzene,
perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene,
and naphthacene.
[0081] The phrase "internal amino acid" means any amino acid in a
peptide sequence that is neither the N-terminal amino acid nor the
C-terminal amino acid.
[0082] The phrase "surface amino acid" means any amino acid that is
exposed to solvent when a protein is folded in its native form.
[0083] The phrase "extracellular signaling protein" means any
protein that is either secreted from a cell, or is tethered to the
outside of a cell, and upon binding to the receptor for that
protein on a target cell triggers a response in the target
cell.
[0084] An "effective amount" of, e.g., a hedgehog polypeptide, with
respect to the subject methods of treatment, refers to an amount of
polypeptide in a preparation which, when applied as part of a
desired dosage regimen brings about, e.g., a change in the rate of
cell proliferation and/or the state of differentiation of a cell
and/or rate of survival of a cell according to clinically
acceptable standards for the disorder to be treated or the cosmetic
purpose.
[0085] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0086] The "growth state" of a cell refers to the rate of
proliferation of the cell and the state of differentiation of the
cell.
[0087] Practice of the present invention will employ, unless
indicated otherwise, conventional techniques of cell biology, cell
culture, molecular biology, microbiology, recombinant DNA, protein
chemistry, and immunology, which are within the skill of the art.
Such techniques are described in the literature.
[0088] II. General Properties of Isolated Hedgehog Proteins
[0089] The polypeptide portion of the hedgehog compositions of the
subject method can be generated by any of a variety of techniques,
including purification of naturally occurring proteins,
recombinantly produced proteins and synthetic chemistry.
Polypeptide forms of the hedgehog therapeutics are preferably
derived from vertebrate hedgehog proteins, e.g., have sequences
corresponding to naturally occurring hedgehog proteins, or
fragments thereof, from vertebrate organisms. However, it will be
appreciated that the hedgehog polypeptide can correspond to a
hedgehog protein (or fragment thereof) which occurs in any metazoan
organism.
[0090] Isolated hedgehog proteins used in the methods of this
invention are naturally occurring or recombinant proteins of the
hedgehog family and may be obtainable from either invertebrate or
from vertebrate sources (see references below). Members of the
vertebrate hedgehog protein family share homology with proteins
encoded by the Drosophila hedgehog (hh) gene (33). To date, the
combined screening of mouse genomic and cDNA libraries has
identified three mammalian hh counterparts referred to as Sonic
hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh),
which also exist in other mammals, including humans, as well as in
fish and in birds. Other members include Moonrat hedgehog (Mhh), as
well as chicken Sonic hh and zebrafish Sonic hh.
[0091] Mouse and chicken Shh and mouse Ihh genes encode
glycoproteins which undergo cleavage, yielding an amino terminal
fragment of about 20 kDa (See FIG. 8) and a carboxy terminal
fragment of about 25 kDa. The most preferred 20 kDa fragment has
the consensus sequence SEQ ID NO: 4 and includes the amino acid
sequences of SEQ ID NOS: 1-3. Various other fragments that
encompass the 20 kDa moiety are considered within the presently
claimed invention. Publications disclosing these sequences, as well
as their chemical and physical properties, include (34-38); PCT
Patent Applications WO 95/23223 (Jessell, Dodd, Roelink and
Edlund), WO 95/18856 (Ingham, McMahon and Tabin) and WO 96/17924
(Beachy et al.).
[0092] Family members useful in the methods of the invention
include any of the naturally-occurring native hedgehog proteins
including allelic, phylogenetic counterparts or other variants
thereof, whether naturally-sourced or produced chemically including
muteins or mutant proteins, as well as recombinant forms and new,
active members of the hedgehog family. Particularly useful hedgehog
polypeptides include SEQ ID NOS: 1-4.
[0093] Isolated hedgehog polypeptides used in the method of the
invention have biological activity. The polypeptides include an
amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99%
homologous to an amino acid sequence from SEQ ID NOS; 1-4. The
polypeptide can also include an amino acid sequence essentially the
same as an amino acid sequence in SEQ ID NOS: 1-4. The polypeptide
is at least 5, 10, 20, 50, 100, or 150 amino acids in length and
includes at least 5, preferably at least 10, more preferably at
least 20, most preferably at least 50, 100, or 150 contiguous amino
acids from SEQ ID NOS: 1-4.
[0094] The preferred polypeptides of the invention include a
hedgehog polypeptide sequence as well as other N-terminal and/or
C-terminal amino acid sequence or it may include all or a fragment
of a hedgehog amino acid sequence. The isolated hedgehog
polypeptide can also be a recombinant fusion protein having a first
hedgehog portion and a second polypeptide portion, e.g., a second
polypeptide portion having an amino acid sequence unrelated to
hedgehog. The second polypeptide portion can be, e.g., histidine
tag, maltose binding protein, glutathione-S-transfera- se, a DNA
binding domain, or a polymerase activating domain.
[0095] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and posttranslational events. The
polypeptide can be made entirely by synthetic means or can be
expressed in systems, e.g., cultured cells, which result in
substantially the same posttranslational modifications present when
the protein is expressed in a native cell, or in systems which
result in the omission of posttranslational modifications present
when expressed in a native cell.
[0096] In a preferred embodiment, isolated hedgehog is a hedgehog
polypeptide with one or more of the following characteristics:
[0097] (i) it has at least 30, 40, 42, 50, 60, 70, 80, 90 or 95%
sequence identity with amino acids of SEQ ID NOS: 1-4;
[0098] (ii) it has a cysteine or a functional equivalent as the
N-terminal end;
[0099] (iii) it may induce alkaline phosphatase activity in
C3H10T1/2 cells;
[0100] (iv) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, or
95%, with a polypeptide of SEQ ID NO; 1-4;
[0101] (v) it can be isolated from natural sources such as
mammalian cells;
[0102] (vi) it can bind or interact with patched; and
[0103] (vii) it is hydrophobically-modified (i.e., it has at least
one hydrophobic moiety attached to the polypeptide).
[0104] III. Other Proteins
[0105] Since techniques exist for engineering a cysteine residue
(or its functional equivalent) into a polypeptide's primary
sequence, virtually any protein can be converted into a
hydrophobically-modified form using the methods described
herein.
[0106] Viral receptors, cell receptors, and cell ligands are useful
because they bind typically to cells or tissues exhibiting many
copies of the receptor. Useful viral-cell protein receptors that
can be complexed together using the methods of this invention
include ICAM1, a rhinovirus receptor; CD2, the Epstein-Barr virus
receptor; and CD4, the receptor for human immunodeficiency virus
(HIV). Other proteins include members of the cell adhesion molecule
family, such as ELAM-1 and VCAM-1 and VCAM-1b and their lymphocyte
counterparts (ligands); the lymphocyte associated antigens LFA1,
LFA2 (CD2) and LFA3 (CD58), CD59 (a second ligand of CD2), members
of the CD11/CD18 family and very late antigens such as VLA4 and
their ligands.
[0107] Immunogens from a variety of pathogens (e.g., from
bacterial, fungal, viral, and other eukaryotic parasites) may also
be used as polypeptides in the methods of the invention. Bacterial
immunogens include, but are not limited to, bacterial sources
responsible for bacterial pneumonia and pneumocystis pneumonia.
Parasitic sources include the Plasmodium malaria parasite. Viral
sources include poxvirus (e.g, cowpox, herpes simplex,
cytomegalovirus); adenoviruses; papovaviruses (e.g.,
papillomavirus); parvoviruses (e.g., adeno-associated virus);
retroviruses (e.g., HTLV I, HTLV II, HIV I and HIV II) and others.
Immunoglobulins, or fragments thereof, may also be polypeptides
that can be modified according to the invention. One can generate
monoclonal Fab fragments recognizing specific antigens using
conventional methods (49) and use the individual Fab domains as
functional moieties in multimeric constructs according to this
invention. Other useful proteins include, gelsolin (50); cytokines,
including the various interferons (interferon-.alpha.,
interferon-.beta., and interferon-.gamma.); the various
interleukins (e.g., IL-1, -2, -3, -4, and the like); the tumor
necrosis factors-.alpha. and -.beta.; monocyte colony stimulating
factor (M-CSF), granulocyte colony stimulating factor (G-CSF),
granulocyte macrophage colony stimulating factor (GM-CSF),
erythropoietin, platelet-derived growth factor (PDGF), and human
and animal hormones, including growth hormone and insulin.
[0108] Generally, the structure of the modified proteins of this
invention has the general formula: A-Cys-[Sp]-B-[Sp]-X, where A is
a hydrophobic moiety; Cys is a cysteine or a functional equivalent
thereof; [Sp] is an optional spacer peptide sequence; B is a
protein (which optionally may have another spacer peptide sequence
as shown); and X is a hydrophobic moiety linked (optionally by way
of the spacer peptide) to the a C-terminal end of the protein or
another surface site of the protein, wherein the derivatized
protein includes at least one of A or X. If X is cholesterol, then
B may, or may not be, a hedgehog protein. As discussed above, the
purpose of the spacer is to provide separation between the
hydrophobic moiety and the rest of the protein so as to make it
easier for the hydrophobic moiety (e.g., a modified N-terminal
cysteine) to link with another moiety which may be a lipid or a
vesicle. The spacer is also intended to make it more difficult for
the modification to interfere with protein function. A spacer may
be as small as a single amino acid in length. Generally, prolines
and glycines are preferred. A particularly preferred spacer
sequence is derived from Sonic hedgehog and consists of the amino
acid sequence: G-P-G-R.
[0109] IV. Production of Recombinant Polypeptides
[0110] The isolated polypeptides described herein can be produced
by any suitable method known in the art. Such methods range from
direct protein synthetic methods to constructing a DNA sequence
encoding isolated polypeptide sequences and expressing those
sequences in a suitable transformed host.
[0111] In one embodiment of a recombinant method, a DNA sequence is
constructed by isolating or synthesizing a DNA sequence encoding a
wild type protein of interest. Optionally, the sequence may be
mutagenized by site-specific mutagenesis to provide functional
analogs thereof. See, e.g., (40) and U.S. Pat. No. 4,588,585.
Another method of constructing a DNA sequence encoding a
polypeptide of interest would be by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides may be
preferably designed based on the amino acid sequence of the desired
polypeptide, and preferably selecting those codons that are favored
in the host cell in which the recombinant polypeptide of interest
will be produced.
[0112] Standard methods may be applied to synthesize an isolated
polynucleotide sequence encoding a isolated polypeptide of
interest. For example, a complete amino acid sequence may be used
to construct a back-translated gene. See Maniatis et al., supra.
Further, a DNA oligomer containing a nucleotide sequence coding for
the particular isolated polypeptide may be synthesized. For
example, several small oligonucleotides coding for portions of the
desired polypeptide may be synthesized and then ligated. The
individual oligonucleotides typically contain 5' or 3' overhangs
for complementary assembly.
[0113] Once assembled (by synthesis, site-directed mutagenesis, or
by another method), the mutant DNA sequences encoding a particular
isolated polypeptide of interest will be inserted into an
expression vector and operatively linked to an expression control
sequence appropriate for expression of the protein in a desired
host. Proper assembly may be confirmed by nucleotide sequencing,
restriction mapping, and expression of a biologically active
polypeptide in a suitable host. As is well known in the art, in
order to obtain high expression levels of a transfected gene in a
host, the gene must be operatively linked to transcriptional and
translational expression control sequences that are functional in
the chosen expression host.
[0114] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations may be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from Esherichia coli, including pCR1, pBR322, pMB9 and
their derivatives, wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages. Preferred E. coli vectors
include pL vectors containing the lambda phage pL promoter (U.S.
Pat. No. 4,874,702), pET vectors containing the T7 polymerase
promoter (Studier et al., Methods in Enzymology 185: 60-89, 1990 1)
and the pSP72 vector (Kaelin et al., supra). Useful expression
vectors for yeast cells, for example, include the 2 T and
centromere plasmids.
[0115] In addition, any of a wide variety of expression control
sequences may be used in these vectors. Such useful expression
control sequences include the expression control sequences
associated with structural genes of the foregoing expression
vectors. Examples of useful expression control sequences include,
for example, the early and late promoters of SV40 or adenovirus,
the lac system, the trp system, the TAC or TRC system, the major
operator and promoter regions of phage lambda, for example pL, the
control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast .alpha.-mating system and other sequences known to control
the expression of genes of prokaryotic or eukaryotic cells and
their viruses, and various combinations thereof.
[0116] Any suitable host may be used to produce in quantity the
isolated hedgehog polypeptides described herein, including
bacteria, fungi (including yeasts), plants, insects, mammals, or
other appropriate animal cells or cell lines, as well as transgenic
animals or plants. More particularly, these hosts may include well
known eukaryotic and prokaryotic hosts, such as strains of E. coli,
Pseudomonas, Bacillus, Streptomyces, fungi, yeast (e.g.,
Hansenula), insect cells such as Spodoptera frugiperda (SF9), and
High Five.TM. (see Example 1), animal cells such as Chinese hamster
ovary (CHO), mouse cells such as NS/O cells, African green monkey
cells COS1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as
well as plant cells.
[0117] It should be understood that not all vectors and expression
control sequences will function equally well to express a given
isolated polypeptide. Neither will all hosts function equally well
with the same expression system. However, one of skill in the art
may make a selection among these vectors, expression control
systems and hosts without undue experimentation. For example, to
produce isolated polypeptide of interest in large-scale animal
culture, the copy number of the expression vector must be
controlled. Amplifiable vectors are well known in the art. See, for
example, (41) and U.S. Pat. Nos. 4,470,461 and 5,122,464.
[0118] Such operative linking of a DNA sequence to an expression
control sequence includes the provision of a translation start
signal in the correct reading frame upstream of the DNA sequence.
If the particular DNA sequence being expressed does not begin with
a methionine, the start signal will result in an additional amino
acid (methionine) being located at the N-terminus of the product.
If a hydrophobic moiety is to be linked to the N-terminal
methionyl-containing protein, the protein may be employed directly
in the compositions of the invention. Neverthless, since the
preferred N-terminal end of the protein is to consist of a cysteine
(or functional equivalent) the methionine must be removed before
use. Methods are available in the art to remove such N-terminal
methionines from polypeptides expressed with them. For example,
certain hosts and fermentation conditions permit removal of
substantially all of the N-terminal methionine in vivo. Other hosts
require in vitro removal of the N-terminal methionine. Such in
vitro and in vivo methods are well known in the art.
[0119] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. For
immunoaffinity chromatography (See Example 1), a protein such as
Sonic hedgehog may be isolated by binding it to an affinity column
comprising of antibodies that were raised against Sonic hedgehog,
or a related protein and were affixed to a stationary support.
Alternatively, affinity tags such as hexahistidine, maltose binding
domain, influenza coat sequence, and glutathione-S-transferase can
be attached to the protein to allow easy purification by passage
over an appropriate affinity column. Isolated proteins can also be
characterized physically using such techniques as proteolysis,
nuclear magnetic resonance, and X-ray crystallography.
[0120] A. Production of Fragments and Analogs
[0121] Fragments of an isolated protein (e.g., fragments of SEQ ID
NOS: 1-4) can also be produced efficiently by recombinant methods,
by proteolytic digestion, or by chemical synthesis using methods
known to those of skill in the art. In recombinant methods,
internal or terminal fragments of a polypeptide can be generated by
removing one or more nucleotides from one end (for a terminal
fragment) or both ends (for an internal fragment) of a DNA sequence
which encodes for the isolated hedgehog polypeptide. Expression of
the mutagenized DNA produces polypeptide fragments. Digestion with
"end nibbling" endonucleases can also generate DNAs which encode an
array of fragments. DNAs which encode fragments of a protein can
also be generated by random shearing, restriction digestion, or a
combination or both. Protein fragments can be generated directly
from intact proteins. Peptides can be cleaved specifically by
proteolytic enzymes, including, but not limited to plasmin,
thrombin, trypsin, chymotrypsin, or pepsin. Each of these enzymes
is specific for the type of peptide bond it attacks. Trypsin
catalyzes the hydrolysis of peptide bonds in which the carbonyl
group is from a basic amino acid, usually arginine or lysine.
Pepsin and chymotrypsin catalyse the hydrolysis of peptide bonds
from aromatic amino acids, such as tryptophan, tyrosine, and
phenylalanine. Alternative sets of cleaved protein fragments are
generated by preventing cleavage at a site which is suceptible to a
proteolytic enzyme. For instance, reaction of the .epsilon.-amino
acid group of lysine with ethyltrifluorothioacetat- e in mildly
basic solution yields blocked amino acid residues whose adjacent
peptide bond is no longer susceptible to hydrolysis by trypsin.
Proteins can be modified to create peptide linkages that are
susceptible to proteolytic enzymes. For instance, alkylation of
cysteine residues with .beta.-haloethylamines yields peptide
linkages that are hydrolyzed by trypsin (51). In addition, chemical
reagents that cleave peptide chains at specific residues can be
used. For example, cyanogen bromide cleaves peptides at methionine
residues (52). Thus, by treating proteins with various combinations
of modifiers, proteolytic enzymes and/or chemical reagents, the
proteins may be divided into fragments of a desired length with no
overlap of the fragments, or divided into overlapping fragments of
a desired length.
[0122] Fragments can also be synthesized chemically using
techniques known in the art such as the Merrifield solid phase F
moc or t-Boc chemistry. Merrifield, Recent Progress in Hormone
Research 23: 451 (1967)
[0123] Examples of prior art methods which allow production and
testing of fragments and 5 analogs are discussed below. These, or
analogous methods may be used to make and screen fragments and
analogs of an isolated polypeptide (e.g., hedgehog) which can be
shown to have biological activity. An exemplary method to test
whether fragments and analogs of hedgehog have biological activity
is found in Example 3.
[0124] B. Production of Altered DNA and Peptide Sequences: Random
Methods
[0125] Amino acid sequence variants of a protein (such as variants
of SEQ ID NOS: 1-4) can be prepared by random mutagenesis of DNA
which encodes the protein or a particular portion thereof. Useful
methods include PCR mutagenesis and saturation mutagenesis. A
library of random amino acid sequence variants can also be
generated by the synthesis of a set of degenerate oligonucleotide
sequences. Methods of generating amino acid sequence variants of a
given protein using altered DNA and peptides are well-known in the
art. The following examples of such methods are not intended to
limit the scope of the present invention, but merely serve to
illustrate representative techniques. Persons having ordinary skill
in the art will recognize that other methods are also useful in
this regard.
[0126] PCR Mutagenesis: Briefly, Taq polymerase (or another
polymerase) is used to introduce random mutations into a cloned
fragment of DNA (42). PCR conditions are chosen so that the
fidelity of DNA synthesis is reduced by Taq DNA polymers using, for
instance, a dGTP/dATP ratio of five and adding Mn.sup.2+ to the PCR
reaction. The pool of amplified DNA fragments is inserted into
appropriate cloning vectors to provide random mutant libraries.
[0127] Saturation Mutagenesis: One method is described generally in
(43). Briefly, the technique includes generation of mutations by
chemical treatment or irradiation of single stranded DNA in vitro,
and synthesis of a cDNA strand. The mutation frequency is modulated
by the severity of the treatment and essentially all possible base
substitutions can be obtained.
[0128] Degenerate Oligonucleotide Mutagenesis: A library of
homologous peptides can be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of degenerate
sequences can by performed in an automatic DNA synthesizer, and the
synthetic genes are then ligated into an appropriate expression
vector. See for example (44, 45) and Itakura et al., Recombinant
DNA, Proc. 3rd Cleveland Symposium on Macromolecules, pp. 273-289
(A. G. Walton, ed.), Elsevier, Amsterdam, 1981.
[0129] C. Production of Altered DNA and Peptide Sequences: Directed
Methods
[0130] Non-random, or directed, mutagenesis provides specific
sequences or mutations in specific portions of a polynucleotide
sequence that encodes an isolated polypeptide, to provide variants
which include deletions, insertions, or substitutions of residues
of the known amino acid sequence of the isolated polypeptide. The
mutation sites may be modified individually or in series, for
instance by: (1) substituting first with conserved amino acids and
then with more radical choices depending on the results achieved;
(2) deleting the target residue; or (3) inserting residues of the
same or a different class adjacent to the located site, or
combinations of options 1-3.
[0131] Clearly, such site-directed methods are one way in which an
N-terminal cysteine (or a functional equivalent) can be introduced
into a given polypeptide sequence to provide the attachment site
for a hydrophobic moiety.
[0132] Alanine scanning Mutagenesis: This method locates those
residues or regions of a desired protein that are preferred
locations for mutagenesis (46). In alanine screening, a residue or
group of target residues are selected and replaced by alanine. This
replacement can affect the interaction of the amino acid with
neighboring amino acids and/or with the surrounding aqueous or
membrane environment. Those having functional sensitivity to the
substitutions are then refined by introducing further or other
variants at, or for, the sites of substitution.
[0133] Oligonucleotide-Mediated Mutagenesis: One version of this
method may be used to prepare substitution, deletion, and insertion
variants of DNA (47). Briefly, the desired DNA is altered by
hybridizing an oligonucleotide primer encoding a DNA mutation to a
DNA template which typically is the single stranded form of a
plasmid or phage containing the unaltered or wild type DNA sequence
template of the desired protein (e.g., the Hedgehog protein). After
hybridization, a DNA polymerase is used to make the second and
complementary strand of DNA of the template that will incorporate
the oligonucleotide primer, and will code for the selected
alteration in the desired DNA sequence. Generally, oligonucleotides
of at least 25 nucleotides in length are used. An optimal
oligonucleotide will have 12 to 15 nucleotides that are completely
complementary to the template on either side of the mutation. This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule.
[0134] Cassette Mutagenesis: This method (48) requires a plasmid or
other vector that contains the protein subunit DNA to be mutated.
The codon(s) in the protein subunit DNA are identified and there is
inserted a unique restriction endonuclease site on each side of the
identified mutation site(s), using the above-described
oligonucleotide-directed mutagenesis method. The plasmid is then
cut at these sites to linearize it. A double-stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
methods. This double-stranded oligonucleotide is the "cassette" and
it has 3' and 5' ends that are compatible with the ends of the
linearized plasmid so that it can be directly ligated therein. The
plasmid now contains the mutated desired protein subunit DNA
sequence.
[0135] Combinatorial Mutagenesis: In one version of this method
(Ladner et al., WO 88/06630), the amino acid sequences for a group
of homologs or other related proteins are aligned, preferably to
promote the highest homology possible. All of the amino acids which
appear at a given position of the aligned sequences can be selected
to create a degenerate set of combinatorial sequences. The
variegated library is generated by combinatorial mutagenesis at the
nucleic acid level, and is encoded by a variegated gene library.
For instance, a mixture of synthetic oligonucleotides can be
ligated enzymically into the gene sequence such that the degenerate
set of potential sequences are expressible as individual peptides,
or alternatively, as a set of proteins containing the entire set of
degenerate sequences.
[0136] D. Other Variants of Isolated Polypeptides
[0137] Included in the invention are isolated molecules that are:
allelic variants, natural mutants, induced mutants, and proteins
encoded by DNA that hybridizes under high or low stringency
conditions to a nucleic acid which encodes a polypeptide such as
the N-terminal fragment of Sonic hedgehog (SEQ ID NO: 1) and
polypeptides bound specifically by antisera to hedgehog peptides,
especially by antisera to an active site or binding site of
hedgehog. All variants described herein are expected to: (i) retain
the biological function of the original protein and (ii) retain the
ability to link to a hydrophobic moiety (e.g, a lipid).
[0138] The methods of the invention also feature uses of fragments,
preferably biologically active fragments, or analogs of an isolated
peptide such as hedgehog. Specifically, a biologically active
fragment or analog is one having any in vivo or in vitro activity
which is characteristic of the peptide shown in SEQ ID NOS: 1-4 or
of other naturally occurring isolated hedgehog. Most preferably,
the hydrophobically-modified fragment or analog has at least 10%,
preferably 40% or greater, or most preferably at least 90% of the
activity of Sonic hedgehog (See Example 3) in any in vivo or in
vitro assay.
[0139] Analogs can differ from naturally occurring isolated protein
in amino acid sequence or in ways that do not involve sequence, or
both. The most preferred polypeptides of the invention have
preferred non-sequence modifications that include in vivo or in
vitro chemical derivatization (e.g., of their N-terminal end), as
well as possible changes in acetylation, methylation,
phosphorylation, amidation, carboxylation, or glycosylation.
[0140] Other analogs include a protein such as Sonic hedgehog or
its biologically active fragments whose sequences differ from the
wild type consensus sequence (e.g., SEQ ID NO: 4) by one or more
conservative amino acid substitutions or by one or more non
conservative amino acid substitutions, or by deletions or
insertions which do not abolish the isolated protein's biological
activity. Conservative substitutions typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following groups:
valine, alanine and glycine; leucine and isoleucine; aspartic acid
and glutamic acid; asparagine and glutamine; serine and threonine;
lysine and arginine; and phenylalanine and tyrosine. The non-polar
hydrophobic amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The positively
charged (basic) amino acids include arginine, lysine, and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Other conservative substitutions
can be readily known by workers of ordinary skill. For example, for
the amino acid alanine, a conservative substitution can be taken
from any one of D-alanine, glycine, beta-alanine, L-cysteine, and
D-cysteine. For lysine, a replacement can be any one of D-lysine,
arginine, D-arginine, homo-arginine, methionine, D-methionine,
ornithine, or D-ornithine.
[0141] Generally, substitutions that may be expected to induce
changes in the functional properties of isolated polypeptides are
those in which: (i) a polar residue, e.g., serine or threonine, is
substituted for (or by) a hydrophobic residue, e.g., leucine,
isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is
substituted for (or by) any other residue (See Example 10); (iii) a
residue having an electropositive side chain, e.g., lysine,
arginine or histidine, is substituted for (or by) a residue having
an electronegative side chain, e.g., glutamic acid or aspartic
acid; or (iv) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having such a
side chain, e.g., glycine.
[0142] Other analogs used within the methods of the invention are
those with modifications which increase peptide stability. Such
analogs may contain, for example, one or more non-peptide bonds
(which replace the peptide bonds) in the peptide sequence. Also
included are: analogs that include residues other than naturally
occurring L-amino acids, such as D-amino acids or non-naturally
occurring or synthetic amino acids such as beta or gamma amino
acids and cyclic analogs. Incorporation of D- instead of L-amino
acids into the isolated hedgehog polypeptide may increase its
resistance to proteases. See, U.S. Pat. No. 5,219,990 supra.
[0143] The term "fragment", as applied to an isolated hedgehog
analog, can be as small as a single amino acid provided that it
retains biological activity. It may be at least about 20 residues,
more typically at least about 40 residues, preferably at least
about 60 residues in length. Fragments can be generated by methods
known to those skilled in the art. The ability of a candidate
fragment to exhibit isolated hedgehog biological activity can be
also assessed by methods known to those skilled in the art as
described herein.
[0144] V. Making Hydrophobic Derivatives
[0145] The inventors have discovered that increasing the overall
hydrophobic nature of a signaling protein, such as a hedgehog
protein, increases the biological activity of the protein. The
potency of a signaling protein such as hedgehog can be increased
by: (a) chemically modifying, such as by adding a hydrophobic
moiety to, the sulfhydryl and/or to the .alpha.-amine of the
N-terminal cysteine (Examples 8 and 9); (b) replacing the
N-terminal cysteine with a hydrophobic amino acid (Example 10); or
(c) replacing the N-terminal cysteine with a different amino acid
and then chemically modifying the substituted residue so as to add
a hydrophobic moiety at the site of the substitution.
[0146] Additionally, modification of a protein such as hedgehog
protein at an internal residue on the surface of the protein with a
hydrophobic moiety by: (a) replacing the internal residue with a
hydrophobic amino acid; or (b) replacing the internal residue with
a different amino acid and then chemically modifying the
substituted residue so as to add a hydrophobic moiety at the site
of the substitution (See Example 10), will retain or enhance the
biological activity of the protein.
[0147] Additionally, modification of a protein such as a hedgehog
protein at the C-terminus with a hydrophobic moiety by: (a)
replacing the C-terminal residue with a hydrophobic amino acid; or
(b) replacing the C-terminal residue with a different amino acid
and then chemically modifying the substituted residue so as to add
a hydrophobic moiety at the site of the substitution, will retain
or enhance the biological activity of the protein.
[0148] There are a wide range of lipophilic moieties with which
hedgehog polypeptides can be derivatived. A lipophilic group can
be, for example, a relatively long chain alkyl or cycloalkyl
(preferably n-alkyl) group having approximately 7 to 30 carbons.
The alkyl group may terminate with a hydroxy or primary amine
"tail". To further illustrate, lipophilic molecules include.
naturally-occurring and synthetic aromatic and non-aromatic
moieties such as fatty acids, esters and alcohols, other lipid
molecules, cage structures such as adamantane and
buckminsterfullerenes, and aromatic hydrocarbons such as benzene,
perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene,
and naphthacene.
[0149] Particularly useful as lipophilic molecules are alicyclic
hydrocarbons, saturated and unsaturated fatty acids and other lipid
and phospholipid moieties, waxes, cholesterol, isoprenoids,
terpenes and polyalicyclic hydrocarbons including adamantane and
buckminsterfullerenes, vitamins, polyethylene glycol or
oligoethylene glycol, (C1-C18)-alkyl phosphate diesters,
--O--CH2--CH(OH)--O--(C12-C18)- -alkyl, and in particular
conjugates with pyrene derivatives. The lipophilic moiety can be a
lipophilic dye suitable for use in the invention include, but are
not limited to, diphenylhexatriene, Nile Red,
N-phenyl-1-naphthylamine, Prodan, Laurodan, Pyrene, Perylene,
rhodamine, rhodamine B, tetramethylrhodamine, Texas Red,
sulforhodamine, 1,1'-didodecyl-3,3,3',3'tetramethylindocarbocyanine
perchlorate, octadecyl rhodamine B and the BODIPY dyes available
from Molecular Probes Inc.
[0150] Other exemplary lipophilic moietites include aliphatic
carbonyl radical groups include 1- or 2-adamantylacetyl,
3-methyladamant-1-ylacety- l, 3-methyl-3-bromo-1-adamantylacetyl,
1-decalinacetyl, camphoracetyl, camphaneacetyl, noradamantylacetyl,
norbornaneacetyl, bicyclo[2.2.2.]-oct-5-eneacetyl,
1-methoxybicyclo[2.2.2.]-oct-5-ene-2-car- bonyl,
cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,
(1R)-(-)-myrtentaneacetyl, 2-norbornaneacetyl,
anti-3-oxo-tricyclo[2.2.1.- 0<2,6>]-heptane-7-carbonyl,
decanoyl, dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or
dodecynoyl.
[0151] Structures of exemplary hydrophobic modifications are shown
in FIG. 12. If an appropriate amino acid is not available at a
specific position, site-directed mutagenesis can be used to place a
reactive amino acid at that site. Reactive amino acids include
cysteine, lysine, histidine, aspartic acid, glutamic acid, serine,
threonine, tyrosine, arginine, methionine, and tryptophan.
Mutagenesis could be used to place the reactive amino acid at the
N- or C-terminus or at an internal position.
[0152] For example, we have discovered that it is possible to
chemically modify an N-terminal cysteine of a biologically active
protein, such as a hedgehog protein, or eliminate the N-terminal
cysteine altogether and still retain the protein's biological
activity, provided that the modified or substituted chemical moiety
is hydrophobic. The inventors have found that enhancement of
hedgehog's biological activity roughly correlates with the
hydrophobicity of the modification. In addition to enhancing the
protein's activity, modifying or replacing the N-terminal cysteine
eliminates unwanted cross reactions and/or modifications of the
cysteine that can occur during production, purification,
formulation, and storage of the protein. The thiol of an N-terminal
cysteine is very reactive due to its proximity to the .alpha.-amine
which lowers the pKa of the cysteine and increases proton
dissociation and formation of the reactive thiolate ion at neutral
or acid pH.
[0153] We have demonstrated that replacement of the N-terminal
cysteine of hedgehog with a hydrophobic amino acid results in a
protein with increased potency in a cell-based signaling assay. By
replacing the cysteine, this approach eliminates the problem of
suppressing other unwanted modifications of the cysteine that can
occur during the production, purification, formulation, and storage
of the protein. The generality of this approach is supported by our
finding that three different hydrophobic amino acids,
phenylalanine, isoleucine, and methionine, each give a more active
form of hedgehog. Therefore, replacement of the cysteine with any
other hydrophobic amino acid should result in an active protein.
Furthermore, since we have found a correlation between the
hydrophobicity of an amino acid or chemical modification and the
potency of the corresponding modified protein in the C3H10T1/2
assay (e.g. Phe>Met, long chain length fatty acids>short
chain length), it could be envisioned that adding more than one
hydrophobic amino acid to the hedgehog sequence would increase the
potency of the protein beyond that achieved with a single amino
acid addition. Indeed, addition of two consecutive isoleucine
residues to the N-terminus of human Sonic hedgehog results in an
increase in potency in the C3H10T1/2 assay as compared to the
mutant with only a single isoleucine added (See Example 10). Thus,
adding hydrophobic amino acids at the N- or C-terminus of a
hedgehog protein, in a surface loop, or some combination of
positions would be expected to give a more active form of the
protein. The substituted amino acid need not be one of the 20
common amino acids. Methods have been reported for substituting
unnatural amino acids at specific sites in proteins (78, 79) and
this would be advantageous if the amino acid was more hydrophobic
in character, resistant to proteolytic attack, or could be used to
further direct the hedgehog protein to a particular site in vivo
that would make its activity more potent or specific. Unnatural
amino acids can be incorporated at specific sites in proteins
during in vitro translation, and progress is being reported in
creating in vivo systems that will allow larger scale production of
such modified proteins.
[0154] It is unexpected that a protein, such as an hedgehog
protein, modified according to the invention, would retain its
biological activity. First, the N-terminal cysteine is conserved in
all known hedgehog protein sequences including fish, frog, insect,
bird, and mammals. Therefore, it is reasonable to expect that the
free sulfhydryl of the N-terminal cysteine is important to the
protein's structure or activity. Second, hedgehog proteins lacking
an N-terminal cysteine, due to proteolytic cleavage or mutation to
hydrophilic amino acids (e.g., aspartic acid or histidine) are
inactive in a the cell-based C3H10T1/2 assay, such as that
described in Example 3.
[0155] There are many modifications of the N-terminal cysteine
which protect the thiol and append a hydrophobic moiety. These
modifications are discussed in more detail below. One of skill in
the art is capable of determining which modification is most
appropriate for a particular therapeutic use. Factors affecting
such a determination include cost and ease of production,
purification and formulation, solubility, stability, potency,
pharmacodynamics and kinetics, safety, immunogenicity, and tissue
targeting.
[0156] A. Chemical Modifications of Primary Amino Acid Sequence
[0157] The chemical modification of the N-terminal cysteine to
protect the thiol, with concomitant activation by a hydrophobic
moiety, can be carried out in numerous ways by someone skilled in
the art. The sulfhydryl moiety, with the thiolate ion as the active
species, is the most reactive functional group in a protein. There
are many reagents that react faster with the thiol than any other
groups. See Chemistry of Protein Conjugation and Cross-Linking (S.
S. Wong, CRC Press, Boca Raton, Fla., 1991). The thiol of an
N-terminal cysteine, such as found in all hedgehog proteins, would
be expected to be more reactive than internal cysteines within the
sequence. This is because the close proximity to the .alpha.-amine
will lower the pKa of the thiol resulting in a greater degree of
proton dissociation to the reactive thiolate ion at neutral or acid
pH. In addition, the cysteine at the N-terminus of the structure is
more likely to be exposed than the other two cysteines in the
hedgehog sequence that are found buried in the protein structure.
We have shown that the N-terminal cysteine is the only amino acid
modified after a 1 h reaction with N-ethylmaleimide at pH 5.5 (See
Example 9), and after a 18 h reaction with N-isopropyliodoacetamide
at pH 7.0 (See Example 9). Other examples of such methods would be
reaction with other .alpha.-haloacetyl compounds, organomercurials,
disulfide reagents, and other N-substituted maleimides. Numerous
hydrophobic derivatives of these active species are available
commercially (e.g., ethyl iodoacetate (Aldrich, Milwaukee Wis.),
phenyl disulfide (Aldrich), and N-pyrenemaleimide (Molecular
Probes, Eugene Oreg.)) or could be synthesized readily (e.g.,
N-alkyliodoacetamides (84), N-alkylmaleimides (85), and
organomercurials (86). We have shown that the N-terminal cysteine
of human Sonic hedgehog can be specifically modified with
N-isopropyliodoacetamide and that the hydrophobically-modified
protein is 2-fold more potent in the C3H10T1/2 assay than the
unmodified protein (See Example 9). It is expected that
modification of Shh with a long-chain alkyl iodoacetamide
derivative will result in a modified protein with even greater
enhancement of potency. Such N-alkyliodoacetamides can be
synthesized readily by ones skilled in the art, using commercially
available starting materials.
[0158] Another aspect to the reactivity of an N-terminal cysteine
is that it can take part in reaction chemistries unique to its
1,2-aminothiol configuration. One example is the reaction with
thioester groups to form an N-terminal amide group via a rapid S to
N shift of the thioester. This reaction chemistry can couple
together synthetic peptides and can be used to add single or
multiple, natural or unnatural, amino acids or other hydrophobic
groups via the appropriately activated peptide. Another example,
demonstrated herein, is the reaction with aldehydes to form the
thiazolidine adduct. Numerous hydrophobic derivatives of thiol
esters (e.g., C2-C24 saturated and unsaturated fatty acyl Coenzyme
A esters (Sigma Chemical Co., St. Louis Mo.)), aldehydes (e.g.,
butyraldehyde, n-decyl aldehyde, and n-myristyl aldehyde
(Aldrich)), and ketones (e.g., 2-, 3-, and 4-decanone (Aldrich))
are available commercially or could be synthesized readily (87,
88). In a similar manner, thiomorpholine derivatives exemplified by
the 1-bromo-2-butanone chemistry described in Example 9 could be
prepared from a variety of .alpha.-haloketone starting materials
(88). Because of the ease of finding alternative routes to
modifying the thiol of the N-terminal cysteine, or any cysteine in
a protein, we do not wish to be bound by the specific examples
demonstrated here.
[0159] The .alpha.-amine of a protein can be modified
preferentially relative to other amines in a protein because its
lower pKa results in higher amounts of the reactive unprotonated
form at neutral or acidic pH. We have shown that modification of
the N-terminal amine with a long chain fatty amide group, while
maintaining a free cysteine thiol group, activates the hedgehog
protein by as much as two orders of magnitude (See Example 8).
Therefore chemistries that can be directed to react preferentially
with the N-terminal amine would be expected to be of use in
increasing the potency of the hedgehog proteins. Aryl halides,
aldehydes and ketones, acid anhydrides, isocyanates,
isothiocyanates, imidoesters, acid halides, N-hydroxysuccinimidyl
(e.g., sulfo-NHS-acetate), nitrophenyl esters, acylimidazoles, and
other activated esters are among those known to react with amine
functions.
[0160] By replacing the N-terminal cysteine of hedgehog with
certain other amino acids, other chemical methods can be used to
add a hydrophobic moiety to the N-terminus. One example is to place
a serine or threonine at the N-terminus, oxidize this amino acid to
form an aldehyde, and then conjugate the protein with a chemical
moiety containing a 1,2 aminothiol structure (e.g., a cysteine). A
second example would be to place a histidine at the N-terminus to
couple to a C-terminal thiocarboxylic acid.
[0161] Chemical Modification of Other Amino Acids.
[0162] There are specific chemical methods for the modification of
many other amino acids. Therefore another route for synthesizing a
more active-form of hedgehog would be to chemically attach a
hydrophobic moiety to an amino acid in hedgehog other than to the
N-terminal cysteine. If an appropriate amino acid is not available
at the desired position, site-directed mutagenesis could be used to
place the reactive amino acid at that site in the hedgehog
structure, whether at the N- or C-terminus or at another position.
Reactive amino acids would include cysteine, lysine, histidine,
aspartic acid, glutamic acid, serine, threonine, tyrosine,
arginine, methionine, and tryptophan. Thus the goal of creating a
more hydrophobic form of hedgehog could be attained by many
chemical means and we do not wish to be restricted by a particular
chemistry or site of modification since our results support the
generality of this approach.
[0163] The hedgehog polypeptide can be linked to the hydrophobic
moiety in a number of ways including by chemical coupling means, or
by genetic engineering.
[0164] To illustrate, there are a large number of chemical
cross-linking agents that are known to those skilled in the art.
For the present invention, the preferred cross-linking agents are
heterobifunctional cross-linkers, which can be used to link the
hedgehog polypeptide and hydrophobic moiety in a stepwise manner.
Heterobifunctional cross-linkers provide the ability to design more
specific coupling methods for conjugating to proteins, thereby
reducing the occurrences of unwanted side reactions such as
homo-protein polymers. A wide variety of heterobifunctional
cross-linkers are known in the art. These include: succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);
N-succinimidyl(4-iodoacetyl)aminobenzoate (SLAB), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB),
1-ethyl-3-(3-dimethylaminopropyl)ca- rbodiimide hydrochloride
(EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-py-
ridyldithio)-tolune (SMPT), N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP), succinimidyl
6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Those
cross-linking agents having N-hydroxysuccinimide moieties can be
obtained as the N-hydroxysulfosuccinimide analogs, which generally
have greater water solubility. In addition, those cross-linking
agents having disulfide bridges within the linking chain can be
synthesized instead as the alkyl derivatives so as to reduce the
amount of linker cleavage in vivo.
[0165] In addition to the heterobifunctional cross-linkers, there
exists a number of other cross-linking agents including
homobifunctional and photoreactive cross-linkers. Disuccinimidyl
suberate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate-2
HCl (DMP) are examples of useful homobifunctional cross-linking
agents, and bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide
(BASED) and
N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate (SANPAH)
are examples of useful photoreactive cross-linkers for use in this
invention. For a recent review of protein coupling techniques, see
Means et al. (1990) Bioconjugate Chemistry 1:2-12, incorporated by
reference herein.
[0166] One particularly useful class of heterobifunctional
cross-linkers, included above, contain the primary amine reactive
group, N-hydroxysuccinimide (NHS), or its water soluble analog
N-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine
epsilon groups) at alkaline pH's are unprotonated and react by
nucleophilic attack on NHS or sulfo-NHS esters. This reaction
results in the formation of an amide bond, and release of NHS or
sulfo-NHS as a by-product.
[0167] Another reactive group useful as part of a
heterobifunctional cross-linker is a thiol reactive group. Common
thiol reactive groups include maleimides, halogens, and pyridyl
disulfides. Maleimides react specifically with free sulfhydryls
(cysteine residues) in minutes, under slightly acidic to neutral
(pH 6.5-7.5) conditions. Halogens (iodoacetyl functions) react with
--SH groups at physiological pH's. Both of these reactive groups
result in the formation of stable thioether bonds.
[0168] The third component of the heterobifunctional cross-linker
is the spacer arm or bridge. The bridge is the structure that
connects the two reactive ends. The most apparent attribute of the
bridge is its effect on steric hindrance. In some instances, a
longer bridge can more easily span the distance necessary to link
two complex biomolecules. For instance, SMPB has a span of 14.5
angstroms.
[0169] Preparing protein-protein conjugates using
heterobifunctional reagents is a two-step process involving the
amine reaction and the sulfhydryl reaction. For the first step, the
amine reaction, the protein chosen should contain a primary amine.
This can be lysine epsilon amines or a primary alpha amine found at
the N-terminus of most proteins. The protein should not contain
free sulfhydryl groups. In cases where both proteins to be
conjugated contain free sulfhydryl groups, one protein can be
modified so that all sulfhydryls are blocked using for instance,
N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,
incorporated by reference herein). Ellman's Reagent can be used to
calculate the quantity of sulfhydryls in a particular protein (see
for example Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and
Riddles et al. (1979) Anal. Biochem. 94:75, incorporated by
reference herein).
[0170] The reaction buffer should be free of extraneous amines and
sulfhydryls. The pH of the reaction buffer should be 7.0-7.5. This
pH range prevents maleimide groups from reacting with amines,
preserving the maleimide group for the second reaction with
sulfhydryls.
[0171] The NHS-ester containing cross-linkers have limited water
solubility. They should be dissolved in a minimal amount of organic
solvent (DMF or DMSO) before introducing the cross-linker into the
reaction mixture. The cross-linker/solvent forms an emulsion which
will allow the reaction to occur.
[0172] The sulfo-NHS ester analogs are more water soluble, and can
be added directly to the reaction buffer. Buffers of high ionic
strength should be avoided, as they have a tendency to "salt out"
the sulfo-NHS esters. To avoid loss of reactivity due to
hydrolysis, the cross-linker is added to the reaction mixture
immediately after dissolving the protein solution.
[0173] The reactions can be more efficient in concentrated protein
solutions. The more alkaline the pH of the reaction mixture, the
faster the rate of reaction. The rate of hydrolysis of the NHS and
sulfo-NHS esters will also increase with increasing pH. Higher
temperatures will increase the reaction rates for both hydrolysis
and acylation.
[0174] Once the reaction is completed, the first protein is now
activated, with a sulfhydryl reactive moiety. The activated protein
may be isolated from the reaction mixture by simple gel filtration
or dialysis. To carry out the second step of the cross-linking, the
sulfhydryl reaction, the lipophilic group chosen for reaction with
maleimides, activated halogens, or pyridyl disulfides must contain
a free sulfhydryl. Alternatively, a primary amine may be modified
with to add a sulfhydryl.
[0175] In all cases, the buffer should be degassed to prevent
oxidation of sulfhydryl groups. EDTA may be added to chelate any
oxidizing metals that may be present in the buffer. Buffers should
be free of any sulfhydryl containing compounds.
[0176] Maleimides react specifically with --SH groups at slightly
acidic to neutral pH ranges (6.5-7.5). A neutral pH is sufficient
for reactions involving halogens and pyridyl disulfides. Under
these conditions, maleimides generally react with --SH groups
within a matter of minutes. Longer reaction times are required for
halogens and pyridyl disulfides.
[0177] The first sulfhydryl reactive-protein prepared in the amine
reaction step is mixed with the sulfhydryl-containing lipophilic
group under the appropriate buffer conditions. The conjugates can
be isolated from the reaction mixture by methods such as gel
filtration or by dialysis.
[0178] Exemplary activated lipophilic moieties for conjugation
include: N-(1-pyrene)maleimide; 2,5-dimethoxystilbene-4'-maleimide,
eosin-5-maleimide; fluorescein-5-maleimide;
N-(4-(6-dimethylamino-2-benzo- furanyl)phenyl)maleimide;
benzophenone-4-maleimide;
4-dimethylaminophenylazophenyl-4'-maleimide (DABMI),
tetramethylrhodamine-5-maleimide, tetramethylrhodamine-6-maleimide,
Rhodamine Red.TM. C2 maleimide, N-(5-aminopentyl)maleimide,
trifluoroacetic acid salt, N-(2-aminoethyl)maleimide,
trifluoroacetic acid salt, Oregon Green.TM. 488 maleimide,
N-(2-((2-(((4-azido-2,3,5,6-te-
trafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide (TFPAM-SS1),
2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl)maleimide
(bisindolylmaleimide; GF 109203X), BODIPY.RTM. FL
N-(2-aminoethyl)maleimi- de,
N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM),
Alexa.TM. 488 C5 maleimide, Alexa.TM. 594 C5 maleimide, sodium
saltN-(1-pyrene)maleimide, 2,5-dimethoxystilbene-4'-maleimide,
eosin-5-maleimide, fluorescein-5-maleimide,
N-(4-(6-dimethylamino-2-benzo- furanyl)phenyl)maleimide,
benzophenone-4-maleimide,
4-dimethylaminophenylazophenyl-4'-maleimide,
1-(2-maleimidylethyl)-4-(5-(-
4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate,
tetramethylrhodamine-5-maleimide, tetramethylrhodamine-6-maleimide,
Rhodamine Red.TM. C2 maleimide, N-(5-aminopentyl)maleimide,
N-(2-aminoethyl)maleimide,
N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoy-
l)amino)ethyl)dithio)ethyl)maleimide,
2-(1-(3-dimethylaminopropyl)-indol-3- -yl)-3-(indol-3-yl)
maleimide, N-(7-dimethylamino-4-methylcoumarin-3-yl)ma- leimide
(DACM), 11H-Benzo[a]fluorene, Benzo[a]pyrene.
[0179] In one embodiment, the hedgehog polypeptide can be
derivatived using pyrene maleimide, which can be purchased from
Molecular Probes (Eugene, Oreg.), e.g., N-(1-pyrene)maleimide or
1-pyrenemethyl iodoacetate (PMIA ester). As illustrated in FIG. 1,
the pyrene-derived hedgehog protein had an activity profile
indicating that it was nearly 2 orders of magnitude more active
than the unmodified form of the protein.
[0180] B. Making Hydrophobic Peptide Derivatives
[0181] According to the invention, the protein can also be modified
using a hydrophobic peptide. As used herein, the term "peptide"
includes a sequence of at least one amino acid residue. Preferably,
the peptide has a length between one amino acid and 18-26 amino
acids, the latter being the typical length of a membrane spanning
segment of a protein. To create a peptide with hydrophobic
character, the amino acids are selected predominantly from the
following hydrophobic amino acids: phenylalanine, isoleucine,
leucine, valine, methionine, tryptophan, alanine, proline, and
tyrosine. The hydrophobic peptide can also contain unnatural amino
acid analogs with hydrophobic character or D-amino acids, peptoid
bonds, N-terminal acetylation or other features that decrease the
peptide's susceptibility to proteolysis. Methods for substituting
unnatural amino acids at specific sites in proteins are known (78,
79).
[0182] Generally, a hydrophobic peptide is appended to various
sites on a protein. One site can be the N-terminal residue.
Alternatively, the hydrophobic peptide is substituted in place of
the N-terminal residue. In another embodiment, a hydrophobic
peptide is appended to the C-terminus of the protein.
Alternatively, the hydrophobic peptide is substituted in place of
the C-terminal residue. The C-terminus can be the native C-terminal
amino acid but it may also be the C-terminus of a truncated protein
so that the hydrophobic peptide is appended to the final C-terminal
amino acid of the truncated form, which is still referred to as the
"C-terminus". A truncated hedgehog protein will retain activity
when up to eleven amino acids are deleted from the native
C-terminal sequence. The hydrophobic peptide may also be inserted
between the N-terminal residue and the internal residue immediately
adjacent to the N-terminal residue, or between the C-terminal
residue and the residue immediately adjacent to the C-terminal
residue, or between two internal residues.
[0183] In certain embodiments, the lipophilic moiety is an
amphipathic polypeptide, such as magainin, cecropin, attacin,
melittin, gramicidin S, alpha-toxin of Staph. aureus, alamethicin
or a synthetic amphipathic polypeptide. Fusogenic coat proteins
from viral particles can also be a convenient source of amphipathic
sequences for the subject hedgehog proteins.
[0184] C. Making Lipid Derivatives
[0185] Another form of protein encompassed by the invention is a
protein derivatized with a variety of lipid moieties. Generally, a
"lipid" is a member of a heterogenous class of hydrophobic
substances characterized by a variable solubility in organic
solvents and insolubility, for the most part, in water. The
principal classes of lipids that are encompassed within this
invention are fatty acids and sterols (e.g., cholesterol).
Derivatized proteins of the invention contain fatty acids which are
cyclic, acyclic (i.e., straight chain), saturated or unsaturated,
monocarboxylic acids. Exemplary saturated fatty acids have the
generic formula: CH.sub.3(CH.sub.2)n COOH. The following table
lists examples of some fatty acids that can be derivatized
conveniently using conventional chemical methods.
[0186] Table 2: Exemplary Saturated and Unsaturated Fatty Acids
Saturated Acids: CH.sub.3(CH.sub.2)n COOH
1 Value of n Common Name 2 butyric acid 4 caproic acid 6 caprylic
acid 8 capric acid 10 lauric acid 12 myristic acid* 14 palmitic
acid* 16 stearic acid* 18 arachidic acid* 20 behenic acid 22
lignoceric acid Unsaturated Acids CH.sub.3CH.dbd.CHCOOH crotonic
acid CH.sub.3(CH.sub.2).sub.3CH.db- d.CH(CH.sub.2).sub.7COOH
myristoleic acid* CH.sub.3(CH.sub.2).sub.-
5CH.dbd.CH(CH.sub.2).sub.7COOH palmitoleic acid*
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7COOH oleic acid*
CH.sub.3(CH.sub.2).sub.3(CH.sub.2CH.dbd.CH).sub.2(CH.sub.2).sub.7COOH
linoleic acid CH.sub.3(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.7CO-
OH linolenic acid CH.sub.3(CH.sub.2).sub.3(CH.sub.2CH.dbd.CH).sub.-
4(CH.sub.2).sub.3COOH arachidonic acid
[0187] The asterisk (*) denotes the fatty acids that we found in
recombinant hedgehog protein secreted from a soluble construct.
[0188] Other lipids that can be attached to the protein include
branched-chain fatty acids and those of the phospholipid group such
as the phosphatidylinositols (i.e., phosphatidylinositol
4-monophosphate and phosphatidylinositol 4,5-biphosphate),
phosphatidycholine, phosphatidylethanolamine, phosphatidylserine,
and isoprenoids such as famesyl or geranyl groups.
[0189] We have demonstrated that lipid-modified hedgehog proteins
can be purified from either a natural source, or can be obtained by
chemically modifying the soluble, unmodified protein. For protein
purified from a natural source, we showed that when full-length
human Sonic hedgehog (Shh) was expressed in insect cells and
membrane-bound Shh purified from the detergent-treated cells using
a combination of SP-Sepharose chromatography and immunoaffinity
chromatography, that the purified protein migrated on reducing
SDS-PAGE gels as a single sharp band with an apparent mass of 20
kDa (See Example 1). The soluble and membrane-bound Shh proteins
were readily distinguishable by reverse phase HPLC, where the
tethered forms eluted later in the acetonitrile gradient (See
Example 1 and FIG. 3). We then demonstrated that human Sonic
hedgehog is tethered to cell membranes in two forms, one form that
contains a cholesterol, and therefore is analogous to the data
reported previously for Drosophila hedgehog (18), and a second
novel form that contains both a cholesterol and a palmitic acid
modification. Soluble and tethered forms of Shh were analyzed by
electrospray mass spectrometry using a triple quadrupole mass
spectrometer, equipped with an electrospray ion source (Example 1)
as well as by liquid chromatography-mass spectrometry (See Example
1). The identity of the N-terminal peptide from endoproteinase
Lys-C digested tethered Shh was confirmed by MALDI PSD mass
spectrometric measurement on a MALDI time of flight mass
spectrometer. The site of palmitoylation was identified through a
combination of peptide mapping and sequence analysis and is at the
N-terminus of the protein (residue 1 of the sequence of the mature
protein in SEQ ID NOS: 1-4). Both tethered forms were equally as
active in the C3H10T1/2 alkaline phosphatase assay, but
interestingly both were about 30-times more potent than soluble
human Shh lacking the tether(s). The lipid modifications did not
significantly affect the apparent binding affinity of Shh for its
receptor, patched (FIG. 7).
[0190] We next tested the utility of the derivatized forms by
assaying the relative potencies of soluble and tethered Shh alone
or in the presence of the anti-hedgehog neutralizing Mab 5E1 on
C3H10T1/2 cells measuring alkaline phosphatase induction. Moreover,
the relative potency of soluble and tethered Shh for binding to
patched was assessed on patched-transfected EBNA-293 cells by FACS
analysis (Example 3).
[0191] For lipid-modified hedgehog obtained by chemically modifying
the soluble, unmodified protein, we have showed that palmitic acid
and other lipids can be added to soluble Shh to create a
lipid-modified forms with increased potency in the C3H10T1/2 assay
(Example 8). We have shown (Examples 1, 2, and 8) that the thiol
and .alpha.-amine on the N-terminal cysteine contribute to the
lipid derivatization reaction. Without wishing to be bound by any
particular theory, lipid modification on proteins starts with the
formation of a thioester intermediate and the lipid moiety is then
transferred to the .alpha.-amine of the N-terminus through the
formation of a cyclic intermediate. Generally, therefore, the
reactive lipid moiety can be in the form of thioesters of saturated
or unsaturated carboxylic acids such as a Coenzyme A thioesters.
Such materials and their derivatives may include, for example,
commercially available Coenzyme A derivatives such as palmitoleoyl
Coenzyme A, arachidoyl Coenzyme A, arachidonoyl Coenzyme A, lauroyl
Coenzyme A and the like. These materials are readily available from
Sigma Chemical Company (St. Louis, Mo., 1998 catalog pp.
303-306).
[0192] The effect of different lipid moieties on functional
activity of hedgehog protein has been assayed (See Example 8 and
FIGS. 10 and 11). Similarly, the effect of different lipid moieties
on functional activity of other proteins such as those described
above in Section III, may be conveniently tested using methods
known to workers of ordinary skill. For instance, functional
testing of gelsolin (50), various interferons (interferon-.alpha.,
interferon-.beta. and interferon-.gamma.), the various interleukins
(e.g., IL-1, -2, -3, -4, and the like), the tumor necrosis
factors-.alpha. and -.beta., and other growth factors that are
lipid-modified according to the invention can be accomplished using
well known methods.
[0193] Although we have established chemical means by which a fatty
acid can be attached to the N-terminal cysteine of hedgehog
proteins, it might be expected that lipids can be attached to the
same or other sites using enzymically catalyzed reactions.
Palmitoylation of proteins in vivo is catalyzed by a class of
enzymes known as palmitoyl-CoA:protein S-palmitoyltransferases.
Using purified enzymes, in vitro acylation of protein substrates
has been demonstrated (80, 81). The substrate specificities of the
palmitoyltransferase enzymes are not well defined; an analysis of
palmitoylation sites of cellular and viral proteins finds little in
the way of a consensus sequence surrounding the modified cysteine
residue, but suggests a common presence of a lysine or arginine
residue within two amino acids of the cysteine, and large,
hydrophobic amino acids near the cysteine. The amino-terminal
sequence of Shh, CGPGRGFG, may fit this consensus sequence and
serve as a recognition site for palmitoylation.
[0194] As an alternative, myristoylation of the amino terminus of
hedgehog proteins could be carried out using an N-myristoyl
transferase (NMT), a number of which have been well characterized
in both mammals (82) and in yeast (83). A recognition site for
N-myristoyltransferase could be engineered into the hedgehog
N-terminal sequence to facilitate recognition by the enzyme. Both
of these strategies would require the use of fatty acyl-coenzyme A
derivatives as substrates, as are used for the non-enzymic fatty
acylation of human Sonic hedgehog described in Example 8.
Alternatively, a protein with an engineered recognition sequence
may be myristoylated during expression in a suitable cell line.
Another method of modifying a protein such as hedgehog with a
hydrophobic moiety is to create a recognition site for the addition
of an isoprenoid group at the C-terminus of the protein. The
recognition site for farnesyl and geranyl-geranyl addition are
known and the protein may be modified during expression in a
eukaryotic cell (Gelb et al., Cur. Opin. Chem. Biol. 2: 40-48
(1998)).
[0195] VI. Multimeric Protein Complexes
[0196] Hydrophobically-modified proteins described herein are
particularly amenable to being made into multimeric protein
complexes. Multimeric protein complexes of the invention include
proteins, optionally attached via their hydrophobic (e.g., lipid)
moiety to a vesicle. The vesicle may be a naturally occurring
biological membrane, purified away from natural material, or the
vesicle may be a synthetic construction. Preferred vesicles are
substantially spherical structures made of amphiphiles, e.g.,
surfactants or phospholipids. The lipids of these spherical
vesicles are generally organized in the form of lipids having one
or more structural layers, e.g., multilamellar vesicles (multiple
onion-like shells of lipid bilayers which encompass an aqueous
volume between the bilayers) or micelles.
[0197] In particular, liposomes are small, spherical vesicles
composed primarily of various types of lipids, phospholipids, and
secondary lipophilic components. These components are normally
arranged in a bilayer formation, similar to the lipid arrangement
of biological membranes.
[0198] Typically, the polar end of a component lipid or lipid-like
molecule is in contact with the surrounding solution, usually an
aqueous solution, while the non-polar, hydrophobic end of the lipid
or lipid-like molecule is in contact with the non-polar,
hydrophobic end of another lipid or lipid-like molecule. The
resulting bilayer membrane (i.e., vesicle) is selectively permeable
to molecules of a certain size, hydrophobicity, shape, and net
charge. Most vesicles are lipid or lipid-like in nature, although
alternative liposome bilayer formulations, comprising a surfactant
with either a lipid or a cholesterol, exist.
[0199] Liposome vesicles may be particularly preferred in that they
find many therapeutic, diagnostic, industrial, and commercial
applications. They are used to deliver molecules which are not
readily soluble in water, or when directed timed release is
desired. Because of their selective permeability to many chemical
compounds, liposomes are useful as delivery vehicles for drugs and
biological materials. Thus, lipid-derivatized proteins such as
hedgehog can be made multimeric by being incorporated into the
lipid bilayer of liposome vesicles. Upon reaching the target site,
the liposomes may be degraded (for example, by enzymes in the
gastro-intestinal tract) or they may fuse with the membranes of
cells.
[0200] Several methods of preparing vesicles such as liposomes are
known. The production of phospholipid vesicles is well known (53).
For a general review of commonly used methods, see (54). Among the
more common of these are (1) sonication of a solution containing
lipids sometimes followed by evaporation/lyophilization and
rehydration (see, e.g. Stryer, Biochemistry, pp. 290-291, Freeman
& Co., New York, (1988), and (55); (2) homogenization of a
lipid solution, sometimes at high pressure or high shearing force
(see e.g. U.S. Pat. No. 4,743,449 issued 10 May 1988, and U.S. Pat.
No. 4,753,788, issued 28 Jun. 1988), (3) hydration and sometimes
sonication of a dried film of vesicle-forming lipids wherein the
lipid film is prepared by evaporation of a solution of lipids
dissolved in an organic solvent (see e.g. U.S. Pat. No. 4,452,747
issued 5 Jun. 1984, U.S. Pat. No. 4,895,719 issued 23 Jan. 1990,
and U.S. Pat. No. 4,946,787 issued 7 Aug. 1990), (4) lyophilization
or evaporation and rehydration (see e.g. U.S. Pat. No. 4,897,355
issued 30 Jan. 1990, EP 267,050 published 5 Nov. 1988, U.S. Pat.
No. 4,776,991 issued 11 Oct. 1988, EP 172,007 published 19 Feb.
1986, and Australian patent application AU-A-48713/85 published 24
Apr. 1986), (5) solvent injection or infusion of a lipid solution
into an aqueous medium or vice versa (see e.g. (56); U.S. Pat. No.
4,921,757 issued 1 May 1990, U.S. Pat. No. 4,781,871 issued 1 Nov.
1988, WO 87/02396 published 24 Mar. 1988, and U.S. Pat. No.
4,895,452 issued 23 Jan. 1990), (6) spray drying (see e.g.
Australian patent application AU-A-48713/85 published 24 Apr. 1986,
and U.S. Pat. No. 4,830,858 issued 16 May 1989), (7) filtration
(see e.g. WO 85/01161), (8) reverse-phase evaporation. See e.g.
(57); and (9) combinations of the above methods. See e.g. (58) and
(59).
[0201] Preferred lipids and lipid-like components suitable for use
in preparing vesicles include phospholipids, a mixture of
phospholipids, polar lipids, neutral lipids, fatty acids, and their
derivatives. A preferred lipid has the characteristic that when
dispersed alone in water, at a temperature above the lipid
transition temperature, they are in a lipid emulsion phase. In
certain embodiments, the lipid is a single-aliphatic chain of
greater than about 12 carbons and can be either saturated or
unsaturated, or substituted in other ways. Suitable lipids include
the ester, alcohol, and acid forms of the following fatty acids:
stearate, oleic acid, linoleic acid, arachidate, arachidonic acid,
and other single-aliphatic chains acids. Further candidates include
the ester, alcohol, and acid forms of the retinols, in particular,
retinol and retinoic acid. Other preferred lipids include
phosphatidylcholine (PC), phosphatidylglycerol (PG) and their
derivatives, created synthetically or derived from a variety of
natural sources.
[0202] In certain embodiments, the vesicle may be stabilized
sterically by the incorporation of polyethylene glycol (PEG), or by
the PEG headgroups of a synthetic phospholipid (PEG conjugated to
distearoyl phosphatidylethanolamine (DSPE), see e.g. (61)).
Preferred surfactants are those with good miscibility such as
Tween.TM., Triton.TM., sodium dodecyl sulfate (SDS), sodium laurel
sulfate, or sodium octylglycoside.
[0203] Preferred surfactants form micelles when added to aqueous
solution above the surfactant's phase transition temperature. The
surfactants may be composed of one or more aliphatic chains. These
aliphatic chains may be saturated, unsaturated, or substituted in
other ways, such as by ethoxylation; typically the aliphatic chain
contains greater than about 12 carbons. Additional suitable
surfactants include the following: lauryl-, myristyl-, linoleyl-,
or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT series (Mona
Industries, Inc., Paterson, N.J.). See also Example 4.
[0204] Preferred sterols and sterol esters suitable for use in
preparing multimeric protein complexes include cholesterol,
cholestanol, cholesterol sulfate, and other cholesterol analogs and
derivatives. The fact that a vesicle may comprise many different
lipids and detergents allows great flexibility in engineering a
tethered protein-vesicle complex with desired properties. For
example, one may produce vesicles that bind different number of
proteins by varying the lipid composition of the starting materials
to create larger vesicles, or by increasing the percentage of
phosphatidylinositol lipids in the vesicle.
[0205] VII. Utilities
[0206] Generally, the modified proteins described herein are useful
for treating the same medical conditions that can be treated with
the unmodified forms of the proteins. However, the
hydrophobically-modified proteins described herein provide several
significant improvements over the unmodified forms. First, their
increased potency enables treatment with smaller amounts of protein
and over shorter periods of time. This will be important in both
systemic and CNS applications. Secondly, replacement of the
N-terminal cysteine with a less chemically reactive amino acid
allows for easier production, formulation, and storage of a protein
for clinical use. Thirdly, the pharmacodynamics of a protein will
be altered by hydrophobic modification and this will allow the
proteins to be localized in the vicinity of the site of
administration, thus increasing their safety, by minimizing
systematic exposure, and their effectiveness by increasing their
local concentration. The proteins of the invention are also useful
in diagnostic compositions and methods to detect their
corresponding receptors.
[0207] As an example of the first point, it has been found that the
half-life of hedgehog is very short after systemic application and
that multiple injections are required to achieve a robust response
to the protein. The higher potency of the modified forms and the
possibility of formulation in liposomes provides a means of
achieving a response with fewer treatments. For CNS applications,
the higher potency provides a means to supply an adequate amount of
protein in the small volumes required for direct injection into the
CNS.
[0208] The importance of the second point is illustrated by the
fact that we have found that the N-terminal cysteine of hedgehog is
highly susceptible to chemical attack, either to form other
chemical adducts or to oxidatively-dimerize with another hedgehog
protein. To prevent this, special buffers and procedures are used
during purification, and dithiothreitol is used in the final
formulation. These precautions necessitate careful evaluation of
the effects of the formulation buffer in animal models.
[0209] As an example of the third point, the more limited the range
over which a protein diffuses away from the site of administration,
the higher the local concentration. This higher local concentration
may therefore allow more specific clinical responses during the
treatment of neurological disorders after direct injection into the
desired region of the brain or spinal cord.
[0210] Similarly, the modified proteins can be administered locally
to the site of bone fractures to help heal these fractures, in the
gonads to treat fertility disorders, intraocularly to treat eye
disorders, and under the skin to treat dermatological conditions,
and to stimulate local hair growth. Localization of the
hydrophobically-modified proteins to the site of administration
therefore reduces possibly undesirable systemic exposure to other
tissues and organs.
[0211] For therapeutic use, hydrophobically-modified proteins of
the invention are placed into pharmaceutically acceptable, sterile,
isotonic formulations and optionally are administered by standard
means well known in the field. The formulation is preferably liquid
or may be lyophilized powder. It is envisioned that a therapeutic
administration of, for instance, a multimeric protein complex may
comprise liposomes incorporating the derivatized proteins described
herein.
[0212] It will be appreciated by persons having ordinary skill in
the art that the particular administration, dosage, and clinical
applications of a hydrophobically-modified protein of the invention
will vary depending upon the particular protein and its biological
activity.
[0213] As but one example of the application of the proteins of
this invention in a therapeutic context, therapeutic
hydrophobically-modified hedgehog proteins can be administered to
patients suffering from a variety of neurological conditions. The
ability of hedgehog protein to regulate neuronal differentiation
during development of the nervous system and also presumably in the
adult state indicates that hydrophobically-modified hedgehog can
reasonably be expected to facilitate control of adult neurons with
regard to maintenance, functional performance, and aging of normal
cells; repair and regeneration processes in lesioned cells; and
prevention of degeneration and premature death which results from
loss of differentiation in certain pathological conditions. In
light of this, the present hydrophobically-modified hedgehog
compositions, by treatment with a local infusion can prevent and/or
reduce the severity of neurological conditions deriving from: (i)
acute, subacute, or chronic injury to the nervous system, including
traumatic injury, chemical injury, vessel injury, and deficits
(such as the ischemia from stroke), together with infectious and
tumor-induced injury; (ii) aging of the nervous system including
Alzheimer's disease; (iii) chronic neurodegenerative diseases of
the nervous system, including Parkinson's disease, Huntington's
chorea, amylotrophic lateral sclerosis and the like; and (iv)
chronic immunological diseases of the nervous system, including
multiple sclerosis. The hydrophobically-modifed protein may also be
injected into the cerebrospinal fluid, e.g., in order to address
deficiencies of brain cells, or into the lymph system or blood
stream as required to target other tissue or organ system-specific
disorders.
[0214] Hedgehog compositions of the invention may be used to
rescue, for example, various neurons from lesion-induced death as
well as guiding reprojection of these neurons after such damage.
Such damage can be attributed to conditions that include, but are
not limited to, CNS trauma infarction, infection, metabolic
disease, nutritional deficiency, and toxic agents (such as
cisplatin treatment). Certain hedgehog proteins cause neoplastic or
hyperplastic transformed cells to become either post-mitotic or
apoptotic. Such compositions may, therefore, be of use in the
treatment of, for instance, malignant gliomas, medulloblastomas and
neuroectodermal tumors.
[0215] The proteins may also be linked to detectable markers, such
as fluoroscopically or radiographically opaque substances, and
administered to a subject to allow imaging of tissues which express
hedgehog receptors. The proteins may also be bound to substances,
such as horseradish peroxidase, which can be used as
immunocytochemical stains to allow visualization of areas of
hedgehog ligand-positive cells on histological sections.
Hydrophobically-modified proteins of the invention, either alone or
as multivalent protein complexes, can be used to specifically
target medical therapies against cancers and tumors which express
the receptor for the protein. Such materials can be made more
effective as cancer therapeutics by using them as delivery vehicles
for antineoplastic drugs, toxins, and cytocidal radionuclides, such
as yttrium 90.
[0216] A toxin may also be conjugated to hydrophobically-modified
hedgehog (or vesicle-containing multivalent complexes thereof) to
selectively target and kill hedgehog-responsive cells, such as a
tumor expressing hedgehog receptor(s). Other toxins are equally
useful, as known to those of skill in the art. Such toxins include,
but are not limited to, Pseudomonas exotoxin, Diphtheria toxin, and
saporin. This approach should prove successful because hedgehog
receptor(s) are expressed in a very limited number of tissues.
Another approach to such medical therapies is to use radioisotope
labeled, hydrophobically-modified protein (or multivalent complexes
thereof). Such radiolabeled compounds will preferentially target
radioactivity to sites in cells expressing the protein receptor(s),
sparing normal tissues. Depending on the radioisotope employed, the
radiation emitted from a radiolabeled protein bound to a tumor cell
may also kill nearby malignant tumor cells that do not express the
protein receptor. A variety of radionuclides may be used.
Radio-iodine (for example, .sup.131I) has been successful when
employed with monoclonal antibodies against CD20 present on B-cell
lymphomas (63).
[0217] The protein compositions to be used in therapy will be
formulated and dosages established in a fashion consistent with
good medical practice taking into account the disorder to be
treated, the condition of the individual patient, the site of
delivery of the isolated polypeptide, the method of administration,
and other factors known to practitioners. The therapeutic may be
prepared for administration by mixing a protein, a
protein-containing vesicle, or a derivatized complex at the desired
degree of purity with physiologically acceptable carriers (i.e.
carriers which are nontoxic to recipients at the dosages and
concentrations employed).
[0218] It is envisioned that local delivery to the site will be the
primary route for therapeutic administration of the proteins of
this invention. Intravenous delivery, or delivery through catheter
or other surgical tubing may also be envisioned. Alternative routes
include tablets and the like, commercially available nebulizers for
liquid formulations, and inhalation of lyophilized or aerosolized
formulations. Liquid formulations may be utilized after
reconstitution from powder formulations.
[0219] The dose administered will be dependent upon the properties
of the vesicle and protein employed, e.g. its binding activity and
in vivo plasma half-life, the concentration of the vesicle and
protein in the formulation, the administration route, the site and
rate of dosage, the clinical tolerance of the patient involved, the
pathological condition afflicting the patient and the like, as is
well known within the skill of the physician. Generally, doses of
from about 5.times.10.sup.-7 to 5.times.10.sup.-9 Molar of protein
per patient per administration are preferred, although the dosage
will depend on the nature of the protein. Different dosages may be
utilized during a series of sequential administrations.
[0220] The invention is also directed towards a pharmaceutical
formulation which includes a hedgehog protein modified according to
the invention in combination with a pharmaceutically acceptable
carrier. In one embodiment, the formulation also includes
vesicles.
[0221] The hydrophobically-modified hedgehog proteins of the
invention are also useful in gene therapy methods.
[0222] For neurodegenerative disorders, several animal models are
available that are believed to have some clinical predicative
value. For Parkinson's disease, models involve the protection, or
the recovery in rodents or primates in which the nigral-striatal
dopaminergic pathway is damaged either by the systemic
administration of MPTP or the local (intracranial) administration
of 6-hydroxydopamine [6-OHDA], two selective dopaminergic toxins.
Specific models are: MPTP-treated mouse model (64); MPTP-treated
primate (marmoset or Rhesus) model (65), and the unilateral 6-OHDA
lesion rat model (66). For ALS, (Amyotrophic lateral sclerosis)
models involve treatment of several mice strains that show
spontaneous motor neuron degeneration, including the wobbler (67)
and pmn mice (68), and of transgenic mice expressing the human
mutated superoxidase dismutase (hSOD) gene that has been linked to
familial ALS (69). For spinal cord injury, the most common models
involve contusion injury to rats, either through a calibrated
weight drop, or fluid (hydrodynamic) injury (70). For Huntington's.
models involve protection from excitotoxin (NMDA, quinolinic acid,
kainic acid, 3-nitro-propionic acid, APMA) lesion to the striatum
in rats (71, 72). Recently, a model of transgenic mice
overexpressing the human trinucleotide expanded repeat in the
huntingtin gene has also been described (73). For multiple
sclerosis, EAE in mice and rats is induced by immunization with MBP
(myelin basic protein), or passive transfer of T cells activated
with MBP (74). For Alzheimer's, a relevant murine model is a
determination of protection against lesion of the fimbria-fornix in
rats (septal lesion), the main nerve bundle supplying the
cholinergic innervation of the hippocampus (75), as well as use of
transgenic mice overexpressing the human beta-amyloid gene. For
peripheral neuropathies a relevant model is protection against loss
of peripheral nerve conductance caused by chemtherapeutic agents
such as taxol, vincristine, and cisplatin in mice and rats
(76).
[0223] This invention will now be described more fully with
reference to the following, non-limiting, Examples.
EXAMPLE 1
Human Sonic Hedgehog is Lipid-Modified when Expressed in Insect
Cells
[0224] A. Expression of Human Sonic Hedgehog
[0225] The cDNA for fill-length human Sonic hedgehog (Shh) was
provided as a 1.6 kb EcoRI fragment subcloned into pBluescript
SK.sup.+ (20) (a gift of David Bumcrot from Ontogeny, Inc.,
Cambridge Mass.). The 5' and 3' NotI sites immediately flanking the
Shh open reading frame were added by unique site elimination
mutagenesis using a Pharmacia kit following the manufacturer's
recommended protocol. The 1.4 kb NotI fragment carrying the
full-length Shh cDNA was then subcloned into the insect expression
vector, pFastBac (Life Technologies, Inc.). Recombinant baculovirus
was generated using the procedures supplied by Life Technologies,
Inc. The resulting virus was used to create a high titer virus
stock. Methods used for production and purification of Shh are
described below. The presence of membrane-associated Shh was
examined by FACS and by Western blot analysis. Peak expression
occurred 48 h post-infection. For Western blot analysis,
supernatants and cell lysates from Shh-infected or uninfected cells
were subjected to SDS-PAGE on a 10-20% gradient gel under reducing
conditions, transferred electrophoretically to nitrocellulose, and
the Shh detected with a rabbit polyclonal antiserum raised against
an N-terminal Shh 15-mer peptide-keyhole limpet hemocyanin
conjugate. The cell lysates were made by incubating the cells for 5
min at 25.degree. C. in 20 mM Na.sub.2HPO.sub.4 pH 6.5, 1% Nonidet
P40 and 150 mM NaCl or 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.5%
Nonidet P40 and 0.5% sodium deoxycholate and then pelleting
particulates at 13,000 rpm for 10 min at 4.degree. C. in an
Eppendorf centrifuge.
[0226] B. Purification of Membrane-Tethered Human Sonic
Hedgehog
[0227] The membrane-tethered form of Shh was produced in High
Five.TM. insect cells (Invitrogen) using the recombinant
baculovirus encoding full-length Shh discussed above. High Five.TM.
cells were grown at 28.degree. C. in sf900 II serum free medium
(Life Technologies, Inc.) in a 10 L bioreactor controlled for
oxygen. The cells were infected in late log phase at ca.
2.times.10.sup.6 cells/mL with virus at a MOI of 3 and harvested 48
h after infection (cell viability at the time of harvest was
>50%). The cells were collected by centrifugation and washed in
10 mM Na.sub.2HPO.sub.4 pH 6.5, 150 mM NaCl pH plus 0.5 mM PMSF.
The resulting cell pellet (150 g wet weight) was suspended in 1.2 L
of 10 mM Na.sub.2HPO.sub.4 pH 6.5, 150 mM NaCl, 0.5 mM PMSF, 5
.mu.M pepstatin A, 10 .mu.g/mL leupeptin, and 2 .mu.g/mL E64, and
120 mL of a 10% solution of Triton X-100 was then added.
[0228] After a 30 min incubation on ice, particulates were removed
by centrifugation (1500.times.g, 10 min). All subsequent steps were
performed at 4-6.degree. C. The pH of the supernatant was adjusted
to 5.0 with a stock solution of 0.5 M MES pH 5.0 (50 mM final) and
loaded onto a 150 ml SP-Sepharose Fast Flow column (Pharmacia). The
column was washed with 300 mL of 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150
mM NaCl, 0.5 mM PMSF, 0.1% Nonidet P-40, then with 200 mL of 5 mM
Na.sub.2HPO.sub.4 pH 5.5, 300 mM NaCl, 0.1% Nonidet P-40, and bound
hedgehog eluted with 5 mM Na.sub.2HPO.sub.4 pH 5.5, 800 mM NaCl,
0.1% Nonidet P-40.
[0229] The Shh was next subjected to immunoaffinity chromatography
on 5E1-Sepharose resin that was prepared by conjugating 4 mg of
antibody per mL of CNBr activated Sepharose 4B resin. The
SP-Sepharose elution pool was diluted with two volumes of 50 mM
HEPES pH 7.5 and batch loaded onto the 5E1 resin (1 h). The resin
was collected in a column, washed with 10 column volumes of PBS
containing 0.1% hydrogenated Triton X-100 (Calbiochem), and eluted
with 25 mM NaH.sub.2PO.sub.4 pH 3.0, 200 mM NaCl, 0.1% hydrogenated
Triton X-100. The elution fractions were neutralized with 0.1
volume of 1M HEPES pH 7.5 and analyzed for total protein content
from absorbance measurements at 240-340 nm and for purity by
SDS-PAGE. Fractions were stored at -70.degree. C.
[0230] Peak fractions from three affinity steps were pooled,
diluted with 1.3 volumes of 50 mM HEPES pH 7.5, 0.2% hydrogenated
Triton X-100 and again batch loaded onto the 5E1 resin. The resin
was collected in a column, washed with three column volumes of PBS
pH 7.2, 1% octylglucoside (US Biochemical Corp.), and eluted with
25 mM NaH.sub.2PO.sub.4 pH 3.0, 200 mM NaCl, 1% octylglucoside. The
elution fractions were neutralized and analyzed as described above,
pooled, filtered through a 0.2 micron filter, aliquoted, and stored
at -70.degree. C.
[0231] When full-length human sonic hedgehog (Shh) was expressed in
High Five.TM. insect cells, over 95% of the N-terminal fragment was
detected by Western blotting in a form that was cell-associated. By
SDS-PAGE, the purified protein migrated as a single sharp band with
apparent mass of 20 kDa (FIG. 1, lane c). The protein migrated
faster by about 0.5 kDa than a soluble version of the protein that
had been produced in E. coli (FIG. 1, lanes b-d), consistent with
data published previously (19). Similarly as described (19), the
soluble and membrane-bound Shh proteins were also readily
distinguishable by reverse phase HPLC where the tethered form
eluted later in the acetonitrile gradient. The concentration of
acetonitrile needed for elution of the membrane-bound form was 60%
versus only 45% with the soluble form, indicating a significant
increase in the hydrophobicity of the protein.
[0232] C. Mass Spectrometry Analysis of Membrane-Tethered Human
Sonic Hedgehog
[0233] Aliquots of Shh were subjected to reverse phase HPLC on a
C.sub.4 column (Vydac, Cat. No. 214TP104, column dimensions 4.6 mm
internal diameter.times.250 mm) at ambient temperature. Bound
components were eluted with a 30 min 0-80% gradient of acetonitrile
in 0.1% trifluoroacetic acid at a flow rate of 1.4 mL/min. The
column effluent was monitored at 280 nm and 0.5 min fractions were
collected. 25 .mu.L aliquots of fractions containing protein were
dried in a Speed Vac concentrator, dissolved in electrophoresis
sample buffer, and analyzed by SDS-PAGE. Hedgehog-containing
fractions were pooled, concentrated 4-fold in a Speed Vac
concentrator and the protein content assayed by absorbance at 280
nm using an extinction coefficient of 1.33 for a 1 mg/mL solution
of Shh. Samples were subjected to ESI-MS on a Micromass Quattro II
triple quadrupole mass spectrometer, equipped with an electrospray
ion source. A volume of 6 .mu.L of HPLC-purified hedgehog was
infused directly into the ion source at a rate of 10 .mu.L/min
using 50% water, 50% acetonitrile, 0.1% formic acid as the solvent
in the syringe pump. Scans were acquired throughout the sample
infusion. All electrospray mass spectral data were acquired and
stored in profile mode and were processed using the Micromass
MassLynx data system.
[0234] Peptides from an endoproteinase Lys-C digest of
pyridylethylated-Shh were analyzed by reverse phase HPLC in line
with the Micromass Quattro II triple quadrupole mass spectrometer.
The digest was separated on a Reliasil C.sub.18 column using a
Michrom.TM. ultrafast Microprotein Analyzer system at a flow rate
of 50 .mu.L/min with a 5-85% acetonitrile gradient in 0.05%
trifluoroacetic acid. Scans were acquired from m/z 400-2000
throughout the run and processed as described above.
[0235] Sequencing of the N-terminal peptide from tethered Shh was
performed by Post Source Decay (PSD)-measurement on a
Voyager-DE.TM. STR (PerSeptive Biosystems, Framingham, Mass.)
time-of-flight (TOF) mass spectrometer using
.alpha.-cyano-4-hydroxycinnamic acid as the matrix (22,23). Exactly
0.5 .mu.L of HPLC-purified endoproteinase Lys-C peptide was mixed
with 0.5 .mu.L of matrix on the target plate. To cover the entire
spectrum of fragment ions, the mirror voltages were decreased from
20 to 1.2 kv in 11 steps.
[0236] Electrospray ionization mass spectrometry data for the
soluble and membrane-bound forms of Shh showed primary species with
masses of 19560 and 20167 Da, respectively (FIG. 2). The measured
mass of 19560 Da matches the predicted mass for Shh starting with
Cys-1 and terminating with Gly-174 (calc. mass of 19560.02 Da). In
contrast, the 20167 Da mass did not agree with any available
prediction nor could the difference in the masses of the tethered
and soluble forms, 607 Da, be accounted for by any known
modification or by aberrant proteolytic processing. Previously,
Porter et al. (18) demonstrated that Drosophila hedgehog contained
a cholesterol moiety and thus it was possible that the mass
difference in the human system was due, at least in part, to
cholesterol (calculated mass for esterified cholesterol is 368.65
Da). The presence of a minor component in the mass spectrum of
tethered Shh at 19796 Da, which differs from the primary peak by
371 Da, supported this notion.
[0237] Further evidence for cholesterol was obtained by treating
the tethered Shh with a mild alkali under conditions that can break
the cholesterol linkage without disrupting peptide bonds (18), and
then reanalyzing the reaction products by mass spectrometry (MS).
Briefly, insect cell-derived Shh was treated with 50 mM KOH, 95%
methanol for 1 h at ambient temperature and then analyzed by ESI-MS
or digested with endoproteinase Lys-C and subjected to LC (liquid
chromatography)-MS on the Micromass Quattro II triple quadrupole
mass spectrometer. For samples subjected to LC-MS, the proteins
were first treated with 4-vinylpyridine. Base treatment shifted the
mass by 387 Da, which is consistent with the loss of cholesterol
plus water (see Table 3). The mass of soluble Shh was not affected
by base treatment. Together, these observations suggested that the
membrane-tethered human Shh contained two modifications, a
cholesterol and a second moiety with a mass of 236 Da. The
similarity in mass between this value and the mass of an added
palmitoyl group (238 Da) suggested that the protein might be
palmitoylated. More accurate estimates of the mass, discussed
below, revealed a correlation within 0.1 Da of the predicted mass
of a palmitoyl moiety.
2TABLE 3 Characterization of tethered Shh by MS. Calculated mass
values were determined using average residue masses in part a and
monoisotopic protonated masses in part b. Mass (Da) Protein
Calculated Measured a. KOH-treated Shh no tether (-treatment)
19560.02 19560 no tether (+treatment) 19560.02 19561 tethered
(-treatment) 20167.14 20167 tethered (+treatment) 19798.49 19780 b.
N-terminal endoproteinase Lys-C peptide (MH.sup.+)* no tether
983.49 983.50 tethered 1221.72 1221.79 *All mass values for
peptides described herein are protonated masses.
[0238] Subsequently, we determined that tethered Shh could be
fractionated into subspecies by HPLC with a modified elution
gradient and we developed a simple HPLC assay for quantifying the
various forms. Results from these analyses are shown in FIG. 3. In
this assay, the unmodified Shh elutes first (peak 1), then
cholesterol-modified Shh elutes (peak 2), and finally product
containing both cholesterol and palmitic acid-modified Shh elutes
(peak 3). The complex shape of peak 3 reflects the presence of a
modified form of the palmitoyl group that was identified through
sequencing by MALDI PSD measurement. The variant was 2 Da smaller
than predicted and may therefore contain an unsaturated bond (data
not shown).
[0239] D. Localization of the Palmitic Acid Modification Within the
Human Sonic Hedgehog Sequence
[0240] The site of palmitoylation within the human sequence was
identified using a combination of peptide mapping and sequence
analysis. FIG. 4B shows results from a peptide mapping analysis of
the soluble protein with an LC-MS readout. Mass data accounting for
over 98% of the soluble Shh sequence could be accounted for from
the analysis. The peak noted with an asterisk corresponds to the
N-terminal peptide (residues 1-9 plus 4-vinylpyridine, observed
mass 983.50 Da, calculated mass 983.49 Da; Table 3). In the
corresponding analysis of the tethered product (FIG. 4A), this
peptide was missing and instead a more hydrophobic peptide with
mass of 1221.79 Da was observed (noted with asterisk).
[0241] The 1221.79 Da moiety is consistent with the presence of a
modified form of the N-terminal peptide, i.e. 983.49 Da for the
peptide component plus 238.23 Da. The 1221.79 Da peptide was next
subjected to sequence analysis by MALDI PSD measurement. The
resulting PSD spectrum is shown in FIG. 5. Ions corresponding to
b1, b2, b4, b5, b8+H.sub.2O, y8, y7, y5, y4, y3, y2, and y1
fragments were detected which confirmed the sequence. In addition,
the b1 and b2 ions indicated that the pyridylethylated Cys-1 adduct
was palmitoylated. Only ions containing Cys-1, contained the added
238.23 Da mass.
[0242] Since cysteine is a normal site of palmitoylation for
proteins in vivo, it was not surprising to find the novel adduct
attached to the N-terminal cysteine. However, two pieces of
evidence suggested that the lipid was attached to the amino group
on the cysteine and not the thiol. First, in the peptide mapping
study, we used 4-vinylpyridine as a spectroscopic tag to monitor
free thiol groups (27). Pyridylethylation is highly specific for
cysteine thiols and adds a 105 Da adduct that can be detected by
MS. The observed Cys-1-containing fragments in the PSD spectrum
contained both palmitoyl and pyridylethyl modifications, implying
the presence of a free thiol group. Second, the tethered Shh was
subjected to automated N-terminal Edman sequencing and no sequence
was obtained, suggesting blockage at the N-terminal .alpha.-amine.
By contrast, the corresponding soluble form of Shh can be sequenced
readily.
EXAMPLE 2
Human Sonic Hedgehog can be Modified with Palmitic Acid in a
Cell-Free System
[0243] Soluble Shh was labeled with .sup.3H-palmitic acid in a
cell-free system using a modified version of a published procedure
(24). A crude microsomal fraction from rat liver was prepared by
subjecting a liver homogenate to sequential centrifugation at
3000.times.g for 10 min, 9000.times.g for 20 min, and
100,000.times.g for 30 min. The 100,000.times.g pellet was
suspended in 10 mM HEPES pH 7.4, 10% sucrose and again centrifuged
at 100,000.times.g for 20 min. The final pellet (derived from 10 g
of liver) was suspended in 3 mL of 20 mM Tris-HCl pH 7.4, 150 mM
NaCl, 1 mM EDTA, 10 .mu.g/mL leupeptin, 0.15% Triton X-100,
aliquoted, and stored at -70.degree. C. Reactions containing 3
.mu.g Shh, 1 .mu.L of rat microsomes, 50 ng/mL Coenzyme A (Sigma),
0.3 mM ATP, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10
.mu.g/mL leupeptin, and 0.5 .mu.Ci-[9,10-.sup.3H]-palmitic acid (50
Ci/mmol; New England Nuclear) were performed at room temperature
for 1 h. Reactions were stopped with reducing electrophoresis
sample buffer, subjected to SDS-PAGE on a 10-20% gradient gel, and
visualized by fluorography.
[0244] As shown in FIG. 1 (lane e), Shh is readily labeled with the
radioactive tracer. None of the ca. hundred other proteins in the
reaction mixture were labeled (see the corresponding Coomassie
blue-stained gel profile in lane j), indicating a high degree of
specificity of the palmitoylation reaction. As further evidence for
the specificity of the palmitoylation reaction, we tested two Shh
variants in which the site of palmitoylation had been eliminated.
FIG. 1 (lane f) shows results from the analysis of a truncated form
of soluble Shh that was lacking the first 10 amino acid residues of
the mature sequence) and lane g, of a mutant form of Shh
containing, at its N-terminal end, a single Cys-1 to Ser point
mutation. Neither of the variants were labeled.
[0245] The significance of the N-terminal cysteine as the site of
lipid derivatization is highlighted by the fact that wild type
soluble Shh is readily labeled while the N-terminal cysteine to
serine mutant is not. The inability to label the N-terminal serine
mutant argues against a simple reaction mechanism where the
palmitoyl moiety is directly attached to the N-terminal
.alpha.-amine since under the test conditions the serine should
have substituted for the cysteine.
[0246] We also tested the role of the free N-terminus using a form
of soluble Shh with an N-terminal histidine (His)-tag extension.
The soluble human Shh used in these studies had been produced
initially as a His-tagged fusion protein with an enterokinase
cleavage site at the junction of the mature sequence and was then
processed with enterokinase to remove the His tag. The His-tagged
Shh was not palmitoylated despite the presence of the free thiol
group of the cysteine (See FIG. 1, lane i). While we cannot rule
out the possibility that the N-terminal extension sterically
inhibits palmitoylation from occurring, Cys-1 is at the P1'
position of the enterokinase cleavage site and is readily
accessible to enzymatic processing. Thus it appears that both the
thiol and .alpha.-amine of Cys-1 contribute to the palmitoylation
reaction. Since all known palmitoylation reactions target the side
chains of Cys, Ser, or Thr residues, we infer that the modification
on hedgehog starts with the formation of a thioester intermediate,
and that the palmitoyl moiety is then transferred to the N-terminus
through the formation of a cyclic intermediate. This hypothesis was
confirmed during studies of the modification of human Sonic
hedgehog using palmityol Coenzyme A (See Example 8).
EXAMPLE 3
Demonstration of Increased Potency of Naturally Ocurring
Fatty-Acylated Human Sonic Hedgehog in a Cell-Based (C3H10T1/2)
Assay
[0247] Shh was tested for function in a cell-based assay measuring
alkaline phosphatase induction in C3H10T1/2 cells (25) with a 5 day
readout. The assay was preformed in a 96-well format. Samples were
run in duplicate. For tethered Shh (100 .mu.g/mL), the samples were
first diluted 200-fold with normal growth medium then subjected to
serial 2-fold dilutions down the plates. Wells were normalized for
potential effects of the added octylglucoside by including 0.005%
octylglucoside in the culture medium. Blocking studies using the
neutralizing murine mAb 5E1 (26) were performed by mixing Shh with
serial dilutions of the antibody for 30 min at ambient temperature
in culture medium prior to adding the test samples to the
plates.
[0248] In this assay, soluble human Shh produces a dose-dependent
response with an IC.sub.50 of 1 .mu.g/mL and a maximal signal at 3
.mu.g/mL (FIG. 6A). Tethered human Shh, with a cholesterol attached
at the C-terminus and a palmitoyl group at the N-terminus,
similarly produced a dose-dependent response in the assay but with
an IC.sub.50 of 0.03 .mu.g/mL and a maximal signal at 0.1 .mu.g/mL,
indicating that it was about 30 times as potent as soluble Shh. To
verify that the observed activity was hedgehog specific, we tested
whether the activity could be inhibited with the anti-hedgehog
neutralizing mAb 5E1. Both soluble and tethered Shh were inhibited
by 5E1 treatment (FIG. 6B). Inhibition of the tethered Shh required
a tenth as much 5E1 consistent with its increased activity in the
assay.
[0249] Tethered Shh was tested in a receptor binding assay,
monitoring its ability to bind patched, using a modified version of
a recently published assay (10). The tethered Shh showed
dose-dependent binding to cells expressing patched with an apparent
IC.sub.50 of 400 ng/mL (FIG. 7). In the same assay, soluble Shh
bound to patched with an apparent IC.sub.50 of 150 ng/mL,
indicating that the tethered form bound only slightly less tightly
to its receptor.
EXAMPLE 4
Analysis of Tethered Human Sonic Hedgehog After Reconstitution Into
Liposomes
[0250] This example illustrates that reconstitution experiments
into positively and negatively charged liposomes by detergent
dilution over a wide range of lipid:protein ratios (w/w) from 1:1
to 100:1 had no effect on tethered Shh activity in the C3H10T1/2
assay.
[0251] Reconstitution into phospholipid-containing liposomes
provides a useful formulation for lipid-containing proteins because
it allows a lipid-containing protein to exist in a near normal
setting. To test whether such a formulation was viable for tethered
Shh we utilized a detergent dilution method to incorporate the
protein into a liposome (60), where preformed liposomes are mixed
with octylglucoside and the protein of interest, and then the
detergent is diluted below its critical micelle concentration, thus
driving the reconstitution. While any of a large number of pure or
lipid mixtures can be utilized, we selected two commercially
available mixtures as models; a negatively charged liposome kit
containing egg L-.alpha.-phosphatidylcholine, dicetyl phosphate,
and cholesterol (Cat. No. L-4262; Sigma, St. Louis, Mo.), and a
positively charged liposome kit consisting of egg phosphatidyl
choline, stearlyamine, and cholesterol (Cat. No L-4137, Sigma).
[0252] Briefly, the lipids were transferred into a Pyrex tube,
dried under a stream of nitrogen, and residual solvent removed by
lyophilization. The lipid was suspended in 10 mM HEPES pH 7.5, 100
mM NaCl, 2.0% octylglucoside, vortexed, and sonicated until the
suspension had turned opalescent in appearance. The lipid was then
filtered through a 0.2 micron filter. Aliquots of tethered Shh,
from baculovirus-infected High Five.TM. insect cells, in
octylglucoside were treated with a 400-, 1000-, 5000-, and a
20000-fold excess of lipid (w/w) and after a 15 min preincubation
the samples were diluted and assayed for activity in the C3H10T1/2
assay.
[0253] Neither the positive nor the negative liposome treatment had
any affect on the activity of the hedgehog indicating that a lipid
carrier was a viable formulation. To verify that the hedgehog
indeed had become reconstituted, parallel samples were subjected to
centrifugation under conditions where the tethered Shh would
normally pellet and the liposomes would float to the surface of the
sample. Under these conditions the tethered Shh floated to the
surface, indicating that reconstitution had occurred.
EXAMPLE 5
Characterization of Membrane-Tethered Human Sonic Hedgehog from
Mammalian (EBNA-293) Cells
[0254] In order to assess whether palmitoylation was a general
modification pathway for Sonic hedgehog or whether it was specific
to insect cell production, the protein was also produced in a
mammalian system in EBNA-293 cells. For expression of full-length
Shh in mammalian cells, the 1.4 kb NotI fragment containing
full-length Shh (See Example 1) was cloned into a derivative of the
vector, CH269 pCEP4 (Invitrogen, San Diego, Calif. (21)). The
construct was transfected into EBNA-293 cells using lipofectamine
(Life Technologies, Inc.) and the cells were harvested 48 h
post-transfection. The expression of surface Shh was verified by
FACS and by Western blot analysis.
[0255] Tethered Shh from EBNA-293 cells was fractionated by reverse
phase HPLC on a narrow bore C.sub.4 column (See FIG. 3). Peaks were
analyzed by ESI-MS (parts a and b of Table 4) or by MALDI-TOF MS on
a Finnigan LaserMat mass spectrometer using
.alpha.-cyano4-hydroxycinnamic acid as the matrix (part c of Table
4). By SDS-PAGE, the protein migrated slightly faster than soluble
Shh, it was retarded on the C.sub.4 column in the reverse phase
HPLC analysis, and, by mass spectrometry, it contained an ion
corresponding to the palmitic acid plus cholesterol modification.
However, unlike the insect cell-derived product where over 80% of
the product contained both the palmitic acid and cholesterol
modification, the HPLC elution profile and data from mass
spectrometry revealed that most of the mammalian cell-derived
protein lacked the palmitoyl moiety (see Table 4 and FIG. 3C). That
is, in peak 2 from EBNA-293 cells the ratio of clipped (des-1-10)
versus intact protein by MS signal was 50% whereas for peak 1 only
about 10% of the Shh was clipped. Interestingly, both the insect
cell and mammalian cell-derived products showed comparable activity
in the C3H10T1/2 assay suggesting that both the cholesterol, and
the cholesterol plus palmitic acid modifications are functional.
Whether the second lipid attachment site is used simply to further
stabilize the association of the protein with membrane or whether
it plays a more active role and affects its conformation or
protein-protein contacts remains to be determined.
[0256] Fatty acid derivatization of proteins is a common post
translational modification that occurs late in the maturation
processes (28,29). For cysteine derivatives, the process is dynamic
involving separate enzymes that add and remove the modification on
the sulfhydryl group. The most common functions of such
derivatization (e.g., palmitoylation) are to alter the
physico-chemical properties of the protein, i.e., to target a
protein to its site of function, to promote protein-protein
interactions, and to mediate protein-membrane interactions (30).
For hedgehog, while the difference in the extent of palmitoylation
in the insect and mammalian cell-derived preparations (80% in
insect cells versus 30% in mammalian cells) was surprising, we do
not know if it is biologically significant or whether it simply
reflects differences in the cellular machinery of the two test
systems for adding and removing palmitic acid. The difference in
the extent of modification in the insect and mammalian cells is
unlikely to be species related since tethered Drosophila hedgehog
that was produced in insect cells lacked palmitic acid (19) despite
having the identical N-terminal sequence.
3TABLE 4 Mass spectrometry analysis of EBNA-293-derived tethered
human Sonic hedgehog. Mass (Da) Protein Calculated Measured a.
bacterial expressed (no tether) 19560.02 19560 b. baculovirus
expressed (tethered) + palmitic acid 19798.49 19796 + palmitic
acid/cholesterol 20167.14 20168 c. EBNA-293 cell expressed
(tethered) peak 1 (9% of total hedgehog) no tether 19560.02 19581
no tether (des 1-9) 18700.02 18712 peak 2 (61% of total hedgehog) +
cholesterol 19928.67 19934 + cholesterol (des 1-10) 18912.48 18889
peak 3 (30% of total hedgehog) + palmitoyl/cholesterol 20167.14
20174
EXAMPLE 6
Lipid Modifications of Rat Sonic Hedgehog
[0257] This Example illustrates that a variety of lipids become
linked to a soluble version of rat Sonic hedgehog when the rat Shh
gene encoding residues 1-174 is expressed in High Five.TM. insect
cells, essentially as for the full-length human Shh described in
Example 1. The lipid modification renders this fraction
membrane-associated. The N-terminal fragment (residues 1-174 of
unprocessed rat Sonic hedgehog) differs by only 2 amino acid
residues to that of the N-terminal fragment of human Sonic
hedgehog. In the rat Sonic hedgehog N-terminal fragment, threonine
replaces serine at position 44, and aspartic acid replaces glycine
at position 173. When rat Sonic hedgehog lacking the autoprocessing
domain is expressed in the High-Five.TM. insect cell/baculovirus
expression system, the majority of the protein is secreted into the
culture medium since it lacks the ability to attach a cholesterol
moiety to the C-terminus. This soluble form has a specific
biological activity (measured by the C3H10T1/2 alkaline phosphatase
induction assay of Example 3) that was similar to that of the
soluble, N-terminal fragment of human Sonic hedgehog expressed and
purified from E. coli.
[0258] However, a small fraction of the total protein remains
associated with the insect cells. The cell-associated rat Sonic
hedgehog protein was purified essentially as described in Example
1, and was found to be significantly more active in the alkaline
phosphatase assay (data not presented) than the soluble, N-terminal
fragments of either human or rat Sonic hedgehog purified from E.
coli and the High-Five.TM. insect cell/baculovirus expression
system, respectively. Subsequent analyses of the rat Sonic hedgehog
N-terminal fragments by HPLC and electrospray mass spectrometry (as
described in Example 1) suggests that the protein is lipid-modified
and that there was more than one type of lipid modification.
Supporting evidence includes the following observations:
[0259] 1. The cell-associated forms elute later than the soluble,
N-terminal fragments of human and rat sonic hedgehog from a C.sub.4
reverse phase HPLC column (Vydac catalog number 214TP104) developed
with a linear 30 min 0-70% acetonitrile gradient in 0.1%
trifluoroacetic acid;
[0260] 2. The masses of the cell-associated forms are consistent
with that expected for the lipid-modified proteins, as shown in
Table 5.
4TABLE 5 Masses of various lipid-modified forms of rat Sonic
hedgehog. Expected Observed Protein Adduct Mass*(MH.sup.+) Mass
(MH.sup.+) unmodified none 19,632.08 19,632 myristoyl-
CH.sub.3(CH.sub.2).sub.12CO-- 19,842.50 19,841 palmitoyl-
CH.sub.3(CH.sub.2).sub.14CO-- 19,870.55 19,868 stearol-
CH.sub.3(CH.sub.2).sub.16CO-- 19,898.60 19,896 arachidoyl-
CH.sub.3(CH.sub.2).sub.18CO-- 19,926.66 19,925 *Average masses were
used in calculating the expected masses
[0261] The location of the lipid moiety was determined using a
combination of sequence analysis and peptide mapping. Automated
N-terminal Edman sequencing of the lipid-modified forms indicated
that the N-terminus was blocked, suggesting that the lipid was
attached to the .alpha.-amine of the N-terminal cysteine.
Endo-Lys-C peptide mapping, MALDI-TOF mass spectrometry and MALDI
PSD analysis (as described in Example 1) of the 4-vinylpyridine
alkylated lipid-modified forms, were used to confirm the location
of the lipid modifications and to determine their exact masses.
[0262] The masses of the N-terminal peptides (residues 1-9
inclusive plus 4-vinylpyridine attached to the thiol side chain of
the N-terminal cysteine) carrying the lipid modifications were
consistent within 0.1 Da with that expected for the lipid-modified
peptides as shown in Table 6.
5TABLE 6 Masses of the N-terminal peptides iosolated from various
lipid-modified forms of rat Sonic hedgehog. Expected Observed
Protein Adduct Mass* (MH.sup.+) Mass (MH.sup.+) myristoyl-
CH.sub.3(CH.sub.2).sub.12CO-- 1193.69 1193.76 palmitoyl-
CH.sub.3(CH.sub.2).sub.14CO-- 1221.72 1221.65 stearoyl-
CH.sub.3(CH.sub.2).sub.16CO-- 1249.75 1249.71 *Monoisotopic masses
were used in calculating the expected masses
[0263] In addition to the lipid-modified peptides shown in Table 6,
peptides with masses of 1191.74, 1219.84 and 1247.82 were also
detected. These masses are consistent with unsaturated forms of
myristate, palmitate and stearate, respectively, although the
position of the double bond in the alkyl chain was not determined.
These observations indicate that both saturated and unsaturated
fatty acids can be attached covalently to the N-terminal cysteine.
For both the saturated and unsaturated lipid-modified peptides,
MALDI PSD analysis as described in Example 1 confirmed that the
lipids were attached covalently to the N-terminal cysteine
residue.
EXAMPLE 7
Lipid Modification of Indian Hedgehog
[0264] To assess whether the palmitoylation reaction was unique to
human Shh or whether it might occur on other hedgehog proteins, we
tested whether human Indian hedgehog (expressed in E. coli as a
His-tagged fusion protein with an enterokinase cleavage site
immediately adjacent to the start of the mature sequence, and
purified exactly as for recombinant human Sonic hedgehog (See
Example 9)) could be palmitoylated using the assay described in
Example 2. Human Indian hedgehog was modified (See FIG. 1, lane h),
indicating that palmitoylation is likely to be a common feature of
hedgehog proteins. The ability to directly label Shh and Ihh with
radioactive palmitic acid in a cell-free system provided a simple
screen for amino acids involved in the modification reaction.
Moreover, Indian hedgehog palmitoylated by the method described in
Example 8 was significantly more potent in the C3H10T1/2 assay than
the unmodified Ihh.
EXAMPLE 8
Lipid Modifications of Sonic Hedgehog Using Acyl-Coenzyme A
[0265] The in vitro acylation of a protein containing an N-terminal
cysteine can be accomplished via a two-step, chemical reaction with
a fatty acid-thioester donor. In the first step, the acyl group of
the thioester donor transfers to the sulfhydryl of the N-terminal
cysteine on the protein by a spontaneous transesterification
reaction. Subsequently, the acyl moiety undergoes a S to N shift to
the .alpha.-amine of the N-terminal cysteine to form a stable amide
bond. Direct acylation of an amine function on a protein may also
occur with prolonged incubation with a thioester, but the presence
of a cysteine on the protein will accelerate the reaction and allow
control over the acylation site. In the present examples,
commercially available Coenzyme A derivatives (Sigma Chemical
Company, St. Louis Mo.) are utilized, but other thioester groups
would also achieve the same result. In fact, certain thioester
leaving groups, such as thiobenzyl esters, would be expected to
react more rapidly. Internal cysteine residues may also promote
acylation to neighboring lysines (i.e., as in an internal
cysteine-lysine pair) and this can be conveniently tested using
synthetic peptides. Secondary acylations occuring on a protein
during reaction with thioesters may be prevented by controlling the
buffer composition, pH, or by site-directed mutagenesis of the
neighboring lysines.
[0266] In preliminary analysis of the effect of acylation on the
ability of human Sonic hedgehog to induce alkaline phosphatase in
C3H10T1/2 cells, reaction mixtures contained 1 mg/mL human Sonic
hedgehog (51 .mu.M), 500 .mu.M of the particular, commercially
available, acyl-Coenzyme A (compounds tested included acetyl-CoA
(C2:0), butyryl-CoA (C4:0), hexanoyl-CoA (C6:0), octanoyl-CoA
(C8:0), decanoyl-CoA (C10:0), lauroyl-CoA (C12:0), myristoyl-CoA
(C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1),
stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), behenoyl-CoA (C22:0),
lignoceroyl-CoA (C24:0), succinyl-CoA, and benzoyl-CoA), 25 mM DTT,
and 50 mM Na.sub.2HPO.sub.4 pH 7.0. The reactions were incubated at
room temperature for 3 h and then analyzed immediately (without
purification) for bioactivity in the C3H10T1/2 assay as described
in Example 3. Samples for analysis by reverse phase HPLC and other
physical methods were usually stored at -70.degree. C. HPLC
analysis was carried out on a Vydac C.sub.4 reverse phase column
(4.6 mm internal diameter.times.250 mm, 5 micron particle) with a
40 min gradient of 5% acetonitrile to 85% acetonitrile in aqueous
0.1% TFA, at a flow rate of 1 mL/min. The effluent was monitored at
280 nm, and fractions were collected in some experiments and
analyzed for hedgehog protein on SDS-PAGE with detection by
Coomassie staining and by Western blotting.
[0267] Comparison of the activity of the various reaction mixtures
(FIG. 10) indicates that a chain length of between 12 and 18
carbons is optimal in inducing high alkaline phosphatase activity
as compared to the unmodified protein. Increasing the chain length
further resulted in an apparent reduction in activity, and the
presence of a double bond in the unsaturated palmitoleoyl-CoA
(C16:1) gave the same activity as the fully saturated palmitoyl-CoA
(C16:0). Upon reverse phase HPLC analysis of the reaction mixtures,
we observed that many of the shorter chain length acyl-CoA
derivatives had not reacted with the hedgehog protein, and
therefore the dependence of biological activity shown in FIG. 10
was not a true reflection of the acyl chain length.
[0268] In order to obtain data on the true activity of the modified
proteins, and on the dependence of activity on acyl chain length,
we developed methods for the synthesis and purification of the
individual N-terminal acylated forms. Palmitoylated, myristoylated,
lauroylated, decanoylated, and octanoylated human Sonic hedgehog
proteins, carrying a single acyl chain attached to the
.alpha.-amine of the N-terminal cysteine, were produced in reaction
mixtures containing 0.80 mg/mL (41 .mu.M) human Sonic hedgehog, 410
.mu.M (10-fold Molar excess) of either palmitoyl-CoA,
myristoyl-CoA, or lauroyl-CoA, or 4.1 mM (100-fold Molar excess) of
either decanoyl-CoA or octanoyl-CoA, 25 mM DTT (for reaction
mixtures containing palmitoyl-CoA, myristoyl-CoA, or lauroyl-CoA)
or 0.5 mM DTT (for reaction mixtures containing decanoyl-CoA or
octanoyl-CoA), and 40 mM Na.sub.2HPO.sub.4 pH 7.0. Reaction
mixtures were incubated at 28.degree. C. for 24 h. Reaction of the
N-terminal cysteine with the acyl thioesters results in the
transfer of the acyl group to the sulfhydryl by a spontaneous
transesterification reaction, which is followed by a S to N shift
to the .alpha.-amine to form a stable amide linkage. The free
sulflhydryl then undergoes a second transesterification reaction,
yielding a protein with a fatty acyl group attached via a thioester
linkage to the sulfhydryl. The thioester-linked acyl group was
removed by adding consecutive 0.11 volume of 1 M Na.sub.2HPO.sub.4
pH 9.0, and 0.11 volume of 1 M hydroxylamine (0.1 M final
concentration) followed by incubation at 28.degree. C. for 18 h,
which leaves only the acyl amide attached to the protein (62). 0.25
volume of 5% octylglucoside was then added (1% final concentration)
and the mixture incubated for 1 h at room temperature. The proteins
were then purified in the presence of 1% octylglucoside using
SP-Sepharose Fast Flow (Pharmacia) and Bio Scale S (Biorad)
cationic ion exchange chromatographies. The purified proteins were
dialyzed against 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 1%
octylglucoside, 0.5 mM DTT, and were stored at -70.degree. C. The
presence of octylglucoside was required to maintain full
solubility; removal of the detergent by dilution and dialysis
resulted in a 75%, 41%, and 15% loss of the palmitoylated,
myristoylated, and lauroylated proteins, respectively. ESI-MS of
the HPLC-purified proteins confirmed their integrity: palmitoylated
Sonic hedgehog, measured mass=19798, calculated mass=19798.43;
myristoylated Sonic hedgehog, measured mass=19770, calculated
mass=19770.33; lauroylated Sonic hedgehog, measured mass=19742,
calculated mass=19742.33; decanoylated Sonic hedgehog, measured
mass=19715, calculated mass=19714.28; octanoylated Sonic hedgehog,
measured mass=19686, calculated mass=19686.23.
[0269] Analysis of the various acylated forms of human Sonic
hedgehog in the C3H10T1/2 assay (FIG. 11) indicated that the
activity of the proteins was dependent upon the chain length. The
palmitoylated, myristoylated, and lauroylated proteins showed
approximately equal activity with EC.sub.50 values of 5-10 ng/mL
(100-200-fold increase in potency as compared to the unmodified
protein). Decanoylated human Sonic hedgehog, with an EC.sub.50
value of 60-70 ng/mL (15-30-fold increase in potency as compared to
the unmodified protein), was less active than the palmitoylated,
myristoylated, and lauroylated forms, while the octanoylated form
was the least active with an EC.sub.50 of 100-200 ng/mL (10-fold
increase in potency as compared to the unmodified protein). All of
the acylated forms were more potent than the unmodified protein
which had an EC.sub.50 of 1000-2000 ng/mL. In addition to the
decrease in EC.sub.50, the palmitoylated, myristoylated, and
lauroylated proteins induced approximately 2-fold more alkaline
phosphatase activity than the unmodified protein, while the
decanoylated and octanoylated proteins induced approximately
1.5-fold more.
[0270] In addition to the increase in potency of the myristoylated
form of human Sonic hedgehog observed in the C3H10T1/2 assay, this
form is significantly more potent than the unmodified protein at
inducing ventral forebrain neurons in explants of embryonic stage
E11 rat brain telencephalon. Incubation of E11 telencephalic
explants with various concentrations of unmodified, or
myristoylated Sonic hedgehog, and subsequent staining of the
explants for the products of the dlx and islet-1/2 genes (markers
of ventral forebrain neurons), indicates that while induction by
the unmodified protein is observed first at 48 nM, induction by the
myristyolated form is observed first at 3 nM. Moreover, while the
unmodified protein induces restricted expression at 3070 nM, the
myristoylated protein induces widespread expression at only 48 nM.
A similar increase in potency was observed when explants of
embryonic stage E9 presumptive telencephalon were incubated with
either the unmodified, or myristoylated proteins. Staining of the
explants for the product of the Nkx2.1 gene (an early marker of
ventral forebrain neurons), indicated that the unmodified protein
induced Nkx2.1 first at 384 nM, while for the myristoylated protein
expression of Nkx2.1 was observed first at 12 nM. Moreover, at 48
nM myristoylated Sonic hedgehog, expression of Nkx2.1 was
widespread while it was undetectable at this concentration using
the unmodified form.
[0271] Additionally, myristoylated human Sonic hedgehog has been
shown to be significantly more protective than the unmodified
protein in reducing the lesion volume which results from
administration of malonate into the striatum of the rat brain (See
Example 16).
EXAMPLE 9
Chemical Derivatives of the N-terminal Cysteine of Human Sonic
Hedgehog
[0272] A. General Methods
[0273] Alkylation of Proteins. Samples containing about 20 .mu.g of
the protein in 50 .mu.L of 6 M guanidine hydrochloride, 50 mM
Na.sub.2HPO.sub.4 pH 7.0, were treated with 0.5 .mu.L of
4-vinylpyridine for 2 h at room temperature. The S-pyridylethylated
protein was precipitated by addition of 40 volumes of cooled
ethanol. The solution was stored at -20.degree. C. for 1 h and then
centrifuged at 14,000.times.g for 8 min at 4.degree. C. The
supernatants were discarded and the precipitate was washed with
cooled ethanol. The protein was stored at -20.degree. C.
[0274] Peptide Mapping. Alkylated protein (0.4 mg/mL in 1 M
guanidine hydrochloride, 20 mM Na.sub.2HPO.sub.4 pH 6.0) was
digested with endo Lys-C (Wako Pure Chemical Industries, Ltd.) at a
1:20 ratio. The digestion was conducted at room temperature for 30
h. The reaction was stopped by acidification with 5 .mu.L of 25%
trifluoroacetic acid. The digest was analyzed on a Waters 2690
Separation Module with a Model 996 photodiode array detector. Prior
to injection, solid guanidine hydrochloride was added into the
digest to a concentration of 6 M to dissolve insoluble material. A
reverse phase Vydac C.sub.18 (2.1 mm internal diameter.times.250
mm) column was used for separation, with a 90 min gradient of 0.1%
trifluoroacetic acid/acetonitrile and 0.1% trifluoroacetic
acid/acetonitrile at a flow rate of 0.2 mL/min. Individual peaks
were collected manually for mass analysis.
[0275] Mass Determination. The molecular masses of intact proteins
were determined by electrospray ionization mass spectroscopy
(ESI-MS) on a Micromass Quattro II triple quadrupole mass
spectrometer. Samples were desalted using an on-line Michrom
Ultrafast Microprotein Analyzer system with a Reliasil C.sub.4 (1
mm internal diameter.times.50 mm) column. The flow rate was 20
.mu.L/min. All electrospray mass spectral data were processed using
the Micromass MassLynx data system. The molecular masses of
peptides were determined by matrix assisted laser desorption
ionization time-of-fight mass spectrometry (MALDI-TOF-MS) on a
Voyager-DE.TM. STR (PerSeptive Biosystems, Framingham, Mass.).
Sequencing of the modifed peptide was performed by Post-source
decay (PSD) measurement on the same instrument.
.alpha.-Cyano-4-hydroxycinnamic acid was used as the matrix.
[0276] N-terminal Sequencing. Proteins were sequenced by Edman
degradation on a Perkin-Elmer Applied Biosystems model 477A
Pulsed-Liquid Protein Sequencer. PTH-thiaproline was made on line
by directly loading thiaproline (thiazolidine-4-carboxylic acid)
into the sample loading cartridge of the sequencer.
[0277] Bacterial expression and purification of wild type soluble
human Sonic hedgehog N-terminal fragment used for chemical
modification. Bacterial pellets from cells expressing Shh at 4-5%
of the total protein were thawed, resuspended in lysis buffer (25
mM Na.sub.2HPO.sub.4 pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5
mM DTT) at a ratio of 1:4 (w/v) and lysed by two passes through a
Gaulin press (mfg. by APV Rannie, Copenhagen, Denmark) at 12,000
p.s.i. All subsequent purification steps were performed at
2-8.degree. C. unless indicated otherwise. The homogenate was
centrifuged at 19,000.times.g for 60 min and MES 0.5 M pH 5, was
added to the resulting lysate at a ratio of 1:10 (v/v). The lysate
(at pH 5.5) was loaded onto an SP Sepharose Fast Flow (Pharmacia,
Piscataway, N.J.) column (4 g E. coli wet weight/mL resin)
equilibrated with 25 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl. The
column was washed with 4 column volumes (CV) of equilibration
buffer, then with 3 CV of 25 mM Na.sub.2HPO.sub.4pH 5.5, 200 mM
NaCl, 0.5 mM DTT, and Histag-Shh was eluted with 800 mM NaCl in the
same buffer. Elution fractions were analyzed for absorbance at 280
nm and by SDS-PAGE. Imidazole (1 M stock solution at pH 7) and NaCl
(5 M stock solution) were added to a pool of the peak Shh
containing fractions from the SP Sepharose eluate to give final
concentrations of 20 mM and 1 M respectively, and this material was
loaded onto a NTA-Ni agarose (Qiagen, Santa Clara, Calif.) column
(20 mg/mL resin) equilibrated with 25 mM Na.sub.2HPO.sub.4 pH 8, 1
M NaCl, 20 mM imidazole, 0.5 mM DTT. The column was washed with 5
CV of the same buffer and Histag-Shh eluted with 3 CV 25 mM
Na.sub.2HPO.sub.4pH 8, 1 M NaCl, 200 mM imidazole, 0.5 mM DTT. The
protein content in the eluate pool from the NTA-Ni column was
determined by absorbance at 280 nm. The pool was warmed to room
temperature and an equal volume of 2.5 M sodium sulfate was added.
The Phenyl Sepharose step was performed at room temperature. The
material was loaded onto a Phenyl Sepharose Fast Flow (Pharmacia,
Piscataway, N.J.) column (20 mg/mL resin) equilibrated in 25 mM
Na.sub.2HPO.sub.4 pH 8, 400 mM NaCl, 1.25 M sodium sulfate, 0.5 mM
DTT. Histag-Shh was eluted with 25 mM Na.sub.2HPO.sub.4 pH 8, 400
mM NaCl, 0.5 mM DTT. Typically, we recovered 2-3 g of His-tagged
Shh from 0.5 kg of bacterial paste (wet weight). The product was
filtered through 0.2 .mu.m filter, aliquoted, and stored at
-70.degree. C. The His-tagged Shh was about 95% pure as determined
by SDS-PAGE. As a further assessment of the characteristics of the
purified product, samples were subjected to evaluation by
electrospray ionization mass spectrometry (ESI-MS). Approximately
50% of the protein was missing the N-terminal methionine.
[0278] To cleave off the hexahistidine tag, enterokinase (Biozyme,
San Diego, Calif.) was incubated with the Histag-Shh at an
enzyme:Shh ratio of 1:1000 (w/w) for 2 h at 28.degree. C. Uncleaved
Histag-Shh and free Histag were removed by passing the digest
through a second NTA-Ni agarose column (20 mg Shh/niL resin). Prior
to loading, imidazole (1 M stock solution at pH 7) and NaCl (5M
stock solution) were added to the digest to give final
concentrations of 20 mM and 600 mM, respectively. This material was
loaded onto a NTA-Ni column equilibrated in 25 mM Na.sub.2HPO.sub.4
pH 8, 600 mM NaCl, 20 mM imidazole, 0.5 mM DTT and the flow through
collected. The column was washed with 1 CV of the same buffer and
pooled with the flow through. MES (0.5 M stock solution at pH 5)
was added to the NTA-Ni agarose unbound fraction to a final
concentration of 50 mM and two volumes of water were added. This
material was loaded onto a second SP Sepharose Fast Flow column (20
mg/mL resin) equilibrated with 5 mM Na.sub.2HPO.sub.4pH 5.5, 150 mM
NaCl, 0.5 mM DTT. The column was washed with 3 CV of equilibration
buffer and 1 CV of the same buffer containing 300 mM NaCl. Shh was
eluted with 5 mM Na.sub.2HPO.sub.4 pH 5.5, 800 mM NaCl, 0.5 mM DTT.
Atomic absorption data revealed that Shh at this stage contained
0.5 mol equivalent of Zn.sup.2+. An equimolar concentration of
ZnCl.sub.2 was added to the Shh eluant and the protein dialyzed
against 5 mM Na.sub.2HPO.sub.4pH 5.5, 150 mM NaCl, 0.5 mM DTT. The
resulting Shh was >98% pure as characterized by SDS-PAGE, size
exclusion chromatography (SEC), and ESI-MS and, by atomic
absorption, contained between 0.9 and 1.1 Zn.sup.2+/Shh.
[0279] ESI-MS data for Histag Shh and products resulting after
removal of the histag are summarized in Table 7.
6TABLE 7 Characterization of Shh by ESI-MS. Mass (Da) Protein
Calculated Measured Histag-Shh (-Met) 21433.82 21434 (Intact)
21565.01 21565 Enterokinase-cleaved Shh 19560.02 19560
[0280] B. Specific Chemical Modifications
[0281] Modification of human Sonic hedgehog with N-ethylmaleimide.
Purified Shh in 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM
DTT was treated with 10 mM N-ethylmaleimide for 1 h on ice and then
dialyzed into 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl. The
MALDI-TOF-MS data showed that the N-ethylmaleimide (NEM)-modified
protein had an increase in mass of 126 Da, which indicates that
only one cysteine residue in Shh was modified by the reagent.
N-terminal sequencing data showed that the protein is sequencible
and that an unusual peak, probably PTH-NEM-Cys related, was
detected at the first cycle (data not shown). Mass spectrometric
analysis of the pyridylethylated-NEM-Shh under denaturing
conditions showed that only two cysteine residues in the protein
were alkylated, confirming that only the thiol group of the
N-terminal cysteine residue was modified by NEM under native
conditions (Table 8). The other two cysteine residues, which are
apparently buried in the hydrophobic core of the protein, cannot be
modified without prior denaturation.
7TABLE 8 Characterization of NEM-modified Shh by MS. Mass Mass
Protein (Calculated) (Measured) Pyridylethylated NEM Shh 19895 Da
if containing 2 free Cys residues 19895 Da if containing 3 free Cys
residues 20000 Da
[0282] When tested in the C3H10T1/2 assay (See Example 3) the
N-ethylmaleimide-modified hedgehog protein was equal in activity to
the unmodified protein. This demonstrates that a free sulfhydryl at
the N-terminus of hedgehog is not required for activity and that
the N-ethylmaleimide moiety is hydrophobic enough to confer some
activity on hedgehog compared to other more hydrophilic
modifications, such as conversion of Cys-1 to His or Asp, which
produce a reduction in activity. Modification of human Sonic
hedgehog with formaldehyde to form an N-terminal thiaproline, and
with acetaldehyde and butyraldehyde to form N-terminal thiaproline
derivatives. For formaldehyde modification, purified Shh at 3 mg/mL
in 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM DTT was
treated with 0.1% formaldehyde, with or without 10% methanol, at
room temperature for 1 to 6 h. The protein was either dialyzed
against 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, or was purified
on a CM-Poros column (Perseptive Biosystems) as described below and
then dilayzed against 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl.
For modification with acetaldehyde or butyraldehyde, purified Shh
at 3 mg/mL in 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM
DTT was treated with 0.1% acetaldehyde or butyraldehyde at room
temperature for 1 h and then the protein purified on a CM-Poros
column. ESI-MS data for the formaldehyde, acetaldehyde-, and
butyraldehyde-treated forms of the protein indicated that their
masses were 13 Da, 27 Da, and 54 Da higher, respectively, than the
unmodified protein (Table 9).
8TABLE 9 Expected and observed masses of human Sonic hedgehog
treated with formaldehyde, acetaldehyde, and butyraldehyde.
Expected mass* Observed mass* Protein (MH.sup.+) (MH.sup.+)
Unmodified 19560.02 19560 Formaldehyde-treated 19572.03 19573
Acetaldehyde-treated 19586.06 19587 Butyraldehyde-treated 19614.11
19614 *Average masses were used in calculating the expected
masses
[0283] For the formaldehyde-treated protein, peptide mapping, as
described above, demonstrated that the site of the modification
occurred in the peptide spanning the first 9 N-terminal residues,
and that the exact mass increase was 12 Da. The results of
MALDI-PSD MS studies of this peptide indicated that the
modification occurred on Cys-1, and could be explained by a
modification of the N-terminal o-amine and the thiol side chain of
Cys-1 to form a thiaproline (See FIG. 12). The structure of the
thiaproline was confirmed by automated N-terminal Edman sequencing
using "on-line" prepared PTH-thiaproline as a standard. For the
acetaldehyde- and butyraldehyde-treated proteins, the ESI-MS data
were consistent with the modifications occuring by means of the
same chemistry as for the reaction with formaldehyde, although the
exact site of modification has not been established. When tested in
the C3H10T1/2 cell-based assay, the formaldehyde-, acetaldehyde-,
and butyraldehyde-modified proteins were approximately 8-fold,
2-fold, and 3-fold, respectively, more potent than unmodified
Shh.
[0284] Modification of human Sonic hedgehog with
N-isopropyliodoacetamide. This example shows that modification of
human Shh with a hydrophobic derivative of iodoacetamide can
enhance the potency of the protein as compared to the unmodified
Shh. Purified Shh (1 mg/mL in 5 mM Na.sub.2HPO.sub.4 pH 7.0, 150 mM
NaCl, 0.1 mM DTT) was incubated with 1 mM N-isopropyliodoacetamide
(NIPIA) at 4.degree. C. for 18 h. DTT was then added to 10 mM final
concentration and the sample was dialyzed extensively against 5 mM
Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM DTT. The sample was
purified on SP Sepharose Fast Flow resin and dialyzed further
against 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM DTT.
ESI-MS data indicated complete conversion to a species with a mass
of 19660, corresponding to the predicted mass value (19659) for the
singly modified protein. Specific modification of the N-terminal
cysteine was confirmed by peptide mapping of proteolytic fragments.
When tested in the C3H10T1/2 cell-based assay, the NIPIA-modified
human Shh was approximately 2-fold more potent than the unmodified
protein. While the modification of the protein resulted in only a
modest increase in potency, it is expected that modification of the
protein with long chain alkyl iodoacetamide derivatives will result
in hydrophobically-modified forms of the protein with much greater
increases in potency, possibly akin to the 100-200-fold increase
observed for the palmitoylated, myristoylated, and lauroylated Shh
proteins (See Example 8).
[0285] Modification of human Sonic hedgehog with 1-bromo-2-butanone
to form a six-membered hydrophobic ring at the N-terminus. A
thiomorpholinyl-(tetrahydrothiazinyl-)derivative of Shh was
prepared by incubating human Shh-N (3 mg/mL in 5 mM
Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.15 mM DTT) with 11 mM
1-bromo-2-butanone at room temperature for 60 min, followed by
reduction with 5 mM NaCNBH.sub.3 at room temperature for 60 min.
The reaction product was purified on a CM-Poros column (Perseptive
Biosystems) as described below and was dialyzed against 5 mM
Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl, 0.5 mM DTT. ESI-MS and
proteolytic peptide mapping data indicated that the product was a
mixture of the expected thiomorpholinyl derivative (calculated
mass=19615, observed mass=19615) and two forms of the protein both
with 16 additional mass units. One of these forms is presumably the
uncyclized keto-thioether intermediate. The mixture was tested in
the C3H10T1/2 assay which indicated that it was approximately
5-fold more potent than the unmodified protein.
EXAMPLE 10
Genetically Engineered Mutations of Human Sonic Hedgehog
[0286] A. Genetically Engineered Mutations of the N-terminal
Cysteine
[0287] In this example, we show that specific replacement of the
N-terminal cysteine of human Sonic hedgehog (Cys-1) by single and
multiple hydrophobic amino acid residues results in increased
potency as compared to the wild type protein in the C3H10T1/2
cell-based assay described in Example 3.
[0288] Construction of Shh Cys-1 mutants. The 584 bp NcoI-XhoI
restriction fragment carrying the His-tagged wild type Shh
N-terminal fragment from p6H-SHH was subcloned into the pUC-derived
cloning vector pNNO5 to construct the plasmid pEAG649. Cys-1
mutants of soluble human Shh were made by unique site elimination
mutagenesis of the pEAG649 plasmid template using a Pharmacia kit
following the manufacturer's recommended protocol. In designing the
mutagenic primers, if a desired mutation did not produce a
restriction site change, a silent mutation producing a restriction
site change was introduced into an adjacent codon to facilitate
identification of mutant clones following mutagenesis. To avoid
aberrant codon usage, substituted codons were selected from those
occurring at least once elsewhere in the human Shh cDNA sequence.
The following mutagenic primers were used: (1) for C1F: 5' GGC GAT
GAC GAT GAC AAA TTC GGA CCG GGC AGG GGG TTC 3' (SEQ ID NO:______),
which introduces an Apol site to make pEAG837; (2) for C1I: 5' GGC
GAT GAC GAT GAC AAA ATA GGA CCG GGC AGG GGG TTC 3' (SEQ ID
NO:______), which loses an RsrII site to make pEAG838; and (3) for
C1M: 5' GGC GAT GAC GAT GAC AAA ATG GGC CCG GGC AGG GGG TTC GGG 3'
(SEQ ID NO:______), which loses both RsrII and AvaII sites to make
pEAG839. Mutations were confirmed by DNA sequencing through a 180
bp NcoI-BgIII restriction fragment carrying the mutant SHH
proteins' N-termini in plasmids pEAG837-839. Expression vectors
were constructed by subcloning each mutant plasmid's 180 bp
NcoI-BgIII fragment and the 404 bp BgIII-XhoI fragment from pEAG649
into the phosphatase-treated 5.64 kb XhoI-NcoI pET11d vector
backbone of p6H-SHH. Presence of the introduced restriction site
change was reconfirmed in the expression vector for each Cys-1
mutant (C1F in pEAG840, C1I in pEAG841, and C1M in pEAG842).
Expression vectors were transformed into competent E. coli
BL21(DE3)pLysS (Stratagene) following the manufacturer's
recommended protocol and selected on LB agar plates containing 100
.mu.g/mL ampicillin and 30 .mu.g/mL chloramphenicol. Individual
colonies were selected and transformed bacteria were grown to an
A.sub.600 of 0.4-0.6 and induced for 3 h with 0.5 mM IPTG.
Bacterial pellets were analyzed for expression of the mutant
proteins by reducing SDS-PAGE and by Western blotting.
[0289] A soluble human Shh mutant with multiple N-terminal
hydrophobic substitutions (C1II) was made by unique site
elimination mutagenesis using a Pharmacia kit following the
manufacturer's recommended protocol. In designing the mutagenic
primers, if a desired mutation did not produce a restriction site
change, a silent mutation producing a restriction site change was
introduced into an adjacent codon to facilitate identification of
mutant clones following mutagenesis. To avoid aberrant codon usage,
substituted codons were selected from those occurring at least once
elsewhere in the human Shh cDNA sequence. The following mutagenic
primer was used on the C1F template plasmid pEAG837 for C1II: 5'
GCG GCG ATG ACG ATG ACA AAA TCA TCG GAC CGG GCA GGG GGT TCG GG 3'
(SEQ ID NO:______), which removes an Apol site to make pEAG872.
Mutations were confirmed by DNA sequencing through a 0.59 kb
NcoI-XhoI restriction fragment carrying the mutant C1II Shh. An
expression vector was constructed by subcloning the mutant
plasmid's NcoI-XhoI fragment into the phosphatase-treated 5.64 kb
XhoI-NcoI pET11d vector backbone of p6H-SHH. Presence of the
introduced restriction site change was reconfirmed in the
expression vector for the C1I mutant, pEAG875. The expression
vector was transformed into competent E. coli BL21(DE3)pLysS
(Stratagene) following the manufacturer's recommended protocol and
selected on LB agar plates containing 100 .mu.g/mL ampicillin and
30 .mu.g/mL chloramphenicol. Individual colonies were selected and
transformed bacteria were grown to an A.sub.600 of 0.4-0.6 and
induced for 3 h with 0.5 mM IPTG. Bacterial pellets were analyzed
as described above to confirm expression of mutant Shh protein.
[0290] Purification of Cys-1 mutants of human Sonic hedgehog. The
His-tagged mutant hedgehog proteins were purified from the
bacterial pellets as described for the wild type protein above
except for two modifications. (1) The Phenyl Sepharose step was
eliminated and instead the protein pool from the first NTA-Ni
agarose column was dialyzed into 25 mM Na.sub.2HPO.sub.4 pH 8, 400
mM NaCl, 0.5 mM DTT in preparation for the enterokinase cleavage
step. (2) The final ion exchange step was changed from step elution
on SP-Sepharose Fast Flow to gradient elution from a CM-Poros
column (Perseptive Biosystems). This was carried out in 50 mM
Na.sub.2HPO.sub.4 pH 6.0 with a 0-800 mM NaCl gradient over 30
column volumes. The pooled peak fractions from this step were
dialyzed into 5 mM Na.sub.2HPO.sub.4 pH 5.5, 150 mM NaCl and were
stored at -80.degree. C. Mass spectrometry of the purified proteins
gave the predicted mass ions for each purified form.
[0291] Activity of the Cvs-1 mutants of human Sonic hedgehog. As
shown in Table 10, mutation of the N-terminal cysteine has a
significant effect on the potency of the resulting hedgehog protein
in the C3H10T1/2 assay. For single changes, potency generally
correlates with the hydrophobicity of the substituted amino acid,
that is phenylalanine and isoleucine give the greatest activation,
methionine is less activating, while histidine and aspartic acid
diminish activity compared to the wild type cysteine. Replacing the
cysteine with two isoleucines gives an additional increase in
activity over the single isoleucine substitution. Given that nine
amino acids are categorized as more hydrophobic than cysteine
(Proteins: structures and molecular properties, 2.sup.nd ed, 1993,
T. E. Creighton, W. H. Freeman Co. page 154), the substitutions
tested above are clearly not an exhaustive survey of the possible
mutations at the N-terminus that can give rise to more active forms
of hedgehog. However, the results demonstrate that activation is
not restricted to a single amino acid structure and that
substitution of more than one amino acid can give a further
increase in potency. Therefore, one skilled in the art could create
forms of hedgehog with other amino acid substitutions at the
N-terminus that would be expected to have greater potency than the
wild type protein.
9TABLE 10 Relative potency of amino acid modifications of human
Sonic hedgehog in the C3H10T1/2 assay. N-TERMINUS RELATIVE POTENCY
C (wild type) 1.times. M 2.times. F 4.times. I 4.times. II
10.times.
[0292] B. Genetically Engineered Mutations of Internal Residues
[0293] Construction of the C1II/A169C mutant. The soluble human Shh
mutant C1/A169C (with cysteine substituted for the dispensable
C-terminal residue A169 which is predicted to have a high
fractional solvent accessibility) was made by unique site
elimination mutagenesis using a Pharmacia kit following the
manufacturer's recommended protocol and employing the mutagenic
oligo design principles described above. The following mutagenic
primer 5' GAG TCA TCA GCC TCC CGA TTT TGC GCA CAC CGA GTT CTC TGC
TTT CAC C 3' (SEQ IDNO:______) was used on C1II Shh template
pEAG872 to add an FspI site to make pSYS049. The C1II/A169C
mutations were confirmed by DNA sequencing through a 0.59 kb
NcoI-XhoI restriction fragment. The expression vector pSYS050 was
constructed by subcloning the NcoI-XhoI fragment into the
phosphatase-treated 5.64 kb XhoI-NcoI pET11d vector backbone of
p6H-SHH. Presence of the introduced restriction site change was
reconfirmed in the expression vector. The expression vector was
transformed into competent E. coli BL21(DE3)pLysS, colonies were
selected, induced, and screened for Shh expression as described
above.
[0294] Purification of the C1II/A169C mutant. The C1II/A169C mutant
was purified as described in Example 9 for wild type Shh except
with the following modifications. (1) EDTA was left out of the
lysis buffer, (2) the order of the NTA-Ni and SP Sepharose steps
were switched and the Phenyl Sepharose step was omitted, (3) after
clarification of the lysed bacteria by centrifugation, additional
NaCl was added to the supernatant to a final concentration of 300
mM, (4) the elution buffer from the NTA-Ni column contained 25 mM
Na.sub.2HPO.sub.4 pH 8.0, 200 mM imidazole, 400 mM NaCl, (5) the
elution pool from the NTA-Ni column was diluted with 3 volumes of
100 mM MES pH 5.0 prior to loading onto the SP Sepharose column,
(6) prior to addition of enterokinase, the SP Sepharose elution
pool was diluted with half a volume of 50 mM Na.sub.2HPO.sub.4 pH
8.0, and (7) the DTT in the elution buffer from the final SP
Sepharose column contained 0.2 mM DTT and the elution pool from
this step was aliquoted and stored at -70.degree. C.
[0295] Hydrophobic modification and activity of the C1II/A169C
mutant. For modification with N-(1-pyrene) maleimide (Sigma),
purified C1II/A169C at 4.6 mg/mL in 5 mM Na.sub.2HPO.sub.4 pH 5.5,
800 mM NaCl, 0.2 mM DTT was diluted with an equal volume of 50 mM
MES pH 6.5 and to this a twentieth of a volume of pyrene maleimide
from a 2.5 mg/mL stock in DMSO was added. The sample was incubated
for 1 h at room temperature in the dark. At this time additional
DTT was added to 0.5 mM and the sample incubated further for an
additional hour at room temperature. The modified protein was
tested directly for activity in the C3H10T1/2 assay as described in
Example 3. Prior to modification, the specific activity of the
protein was EC.sub.50=0.22 .mu.g/mL, while after treatment with
pyrene maleimide the specific activity was increased to
EC.sub.50=0.08 .mu.g/mL. Increases in the specific activity of the
modified product by up to 3-fold were observed frequently
indicating that the addition of the hydrophobic group near the
C-terminus of Shh resulted in a further increase in activity as
compared to the C1II starting material. When compared to the wild
type unmodified Sonic hedgehog protein, the N-(1-pyrene)
maleimide-modified C1II protein was approximately 30-fold more
potent. While pyrene maleimide provided a simple test system for
evaluating modification at this site, other hydrophic maleimides or
other cysteine targeted chemistries can also be used.
EXAMPLE 11
Comparison of the Potency of Various Hydrophobically-Modified Forms
of Human Sonic Hedgehog in the C3H10T1/2 Assay
[0296] The activity of various hydrophobically-modified forms of
human Sonic hedgehog (prepared using the chemistries and genetic
engineeering methods described in Section V) was tested in the
C3H10T1/2 assay as described in Example 3.
[0297] The derivatives were assayed over a concentration range as
described in Example 3. The concentration of hedgehog derivative
that resulted in 50% of the maximum response in the assay was
compared to the wild type concentration. The relative activities
are shown in Table 11, below, and in FIG. 13.
10TABLE 11 Relative Potency of Hedgehog Derivatives in the
C3H10T1/2 assay. EC.sub.50 (x-fold more potent Modification than
wild type Shh) C:16 palmitoyl 100 C:14 myristoyl 100 C:12 lauroyl
100 C:10 decanoyl 33 Isoleucyl-isoleucyl with A169C pyrenyl 30 C:8
octanoyl 10 Isoleucyl-isoleucyl 10 C:0 thiaprolyl 8 Thiomorpholinyl
5 Phenylalanyl 4 Isoleucyl 4 N-isopropylacetamidyl 2 Methionyl 2
N-ethylmaleimidyl 1 Cysteinyl (wild type) 1 Aspartyl <1 Histidyl
<1
[0298] The C3H10T1/2 assay demonstrates that a wide variety of
hydrophobic modifications to hedgehog increase the protein's
activity when compared to the wild type, unmodified protein.
Hydrophilic modifications (aspartic acid and histidine) do not have
this effect.
EXAMPLE 12
Evaluating the Efficacy of Hydrophobically-Modified Human Sonic
Hedgehog in a Rat Malonate-Induced Striatal Lesion Assay
[0299] Injection of malonate, an inhibitor of the mitochondrial
enzyme succinate dehydrogenase, into the rat striatum (the rodent
equivalent of the primate caudate and putamen) causes degeneration
of striatal medium spiny neurons. In humans, degeneration of medium
spiny neurons in the caudate and putamen is the primary
pathological feature of Huntington's disease. Thus, the
malonate-induced striatal lesion in rats can be used as a model to
test whether hydrophobically-modified hedgehog proteins can prevent
the death of the neurons which degenerate in Huntington's
disease.
[0300] Sprague-Dawley rats were injected with various
concentrations of hydrophobically-modified human Sonic hedgehog in
the striatum using stereotaxic techniques. Stereotaxic injections
(2 .mu.L) were performed under sodium pentobarbital anesthesia (40
mg/kg) and placed at the following coordinates: 0.7 mm anterior to
bregma, 2.8 mm lateral to the midline, and 5.5 mm ventral to the
surface of the skull at bregma. At various times (usually 48 h)
after injection of the hydrophobically-modified protein, rats were
anesthetized with isoflurane and given a stereotaxic injection of
malonate (2 .mu.mol in 2 .mu.L) at the same coordinates in the
striatum. Four days after malonate injection, rats were sacrificed
and their brains removed for histological analysis. Coronal
sections were cut through the striatum at a thickness of 25 .mu.m
and stained for cytochrome oxidase activity to distinguish lesioned
from unlesioned tissue. The volume of the lesion in the striatum is
measured using an image analysis system.
[0301] The effect of hydrophobically-modified human Sonic hedgehog
protein in the malonate-induced rat striatal lesion model is shown
in FIG. 14. Unmodified Sonic hedgehog (prepared as described in
Example 9), myristoylated Shh (prepared as described in Example 8),
and the C1II mutant of Shh (prepared as described in Example 10)
all reduced lesion volume to a similar extent in this model.
However, the hydrophobically-modified proteins (myristoylated Shh
and C1II Shh) showed an increase in potency relative to the
unmodified Sonic hedgehog.
EXAMPLE 13
N-Octylmaleimide Derivitization of sHh-N
[0302] For a 1 mg/ml Final Concentration
[0303] 1) Make a 20 mM solution of octylmaleimide (m.w.=209) in
DMSO (.about.4.2 mg/ml).
[0304] 2) Dilute stock of 10 mg/ml sHh-N (in 5 mM NaPO4 pH 5.5, 150
mM NaCl, 0.5 mM DTT) 10-fold with PBS (Gibco product #20012-027, pH
7.2) to give a 1 mg/ml (or 50 uM) sHh-N solution. [NOTE: DTT, which
competes with sHh-N for maleimide in the subsequent reaction, is
also 50 .mu.M in this solution.]
[0305] 3) Immediately add 1/200 vol. of octylmaleimide to the 1
mg/ml sHh-N (i.e. 5 .mu.l/1 ml). This gives a 2:1 molar ratio (100
.mu.M:50 .mu.M) of octylmaleimide to sHh-N.
[0306] 4) Mix this solution by gentle inversion of the tube and
incubate for 1 hour at room temperature.
[0307] 5) Finally, 1/1000 vol. of 0.35 M DTT was added to each tube
to scavenge any remaining octylmaleimide and to serve as a
reductant.
[0308] 6) For a vehicle control, combine a solution of vehicle (5
mM NaPO4 pH 5.5, 150 mM NaCl, 0.5 mM DTT) with PBS (Gibco product
#20012-027, pH 7.2) in a 1:10 ratio. Add 1/400 vol. of 20 mM
octylmaleimide in DMSO and a 1/400 vol. of DMSO to give a final
concentration of 50 .mu.M N-octylmaleimide and 0.5% DMSO. Finally,
add 1:1000 vol. of 0.35 M DTT.
[0309] Approximate Composition of the 1 mg/ml N-octylmaleimide
sHh-N Solution
[0310] PBS (.about.pH 7.2)
[0311] 50 .mu.M sHh-N conjugated to N-octylmaleimide
[0312] 50 .mu.M DTT conjugated to N-octylmaleimide
[0313] 350 .mu.M DTT
[0314] 0.5% DMSO
[0315] Approximate Composition of the N-octylmaleimide Vehicle
Solution
[0316] PBS (.about.pH 7.2)
[0317] 50 .mu.M DTT conjugated to N-octylmaleimide
[0318] 350 .mu.M DTT
[0319] 0.5% DMSO
[0320] For a 3 mg/ml Final Concentration
[0321] 1) Make a 60 mM solution of octylmaleimide (m.w.=209) in
DMSO (.about.12.6 mg/ml).
[0322] 2) Dilute stock of 10 mg/ml sHh-N (in 5 mM NaPO4 pH 5.5, 150
mM NaCl, 0.5 mM DTT) 10-fold with PBS (Gibco product #20012-027, pH
7.2) to give a 3 mg/ml (or 150 uM) sHh-N solution. [NOTE: DTT,
which competes with sHh-N for maleimide in the subsequent reaction,
is also 150 .mu.M in this solution.]
[0323] 3) Immediately add 1/200 vol. of octylmaleimide to the 3
mg/ml sHh-N (i.e. 5 .mu.l/1 ml). This gives a 2:1 molar ratio (300
.mu.M:150 .mu.M) of octylmaleimide to sHh-N.
[0324] 4) Mix this solution by gentle inversion of the tube and
incubate for 1 hour at room temperature.
[0325] 5) Finally, add 1/1000 vol. of 0.35 M DTT to each tube to
scavenge any remaining octylmaleimide and to serve as a
reductant.
[0326] 6) For a vehicle control, combine a solution of vehicle (5
mM NaPO4 pH 5.5, 150 mM NaCl, 0.5 mM DTT) with PBS (Gibco product
#20012-027, pH 7.2) in a 3:7 ratio. Add 1/400 vol. of 60 mM
octylmaleimide in DMSO and a 1/400 vol. of DMSO to give a final
concentration of 150 .mu.M N-octylmaleimide and 0.5% DMSO. Finally,
add 1:1000 vol. of 0.5 M DTT.
[0327] Approximate Composition of the 3mg/ml N-octylmaleimide sHh-N
Solution
[0328] PBS (.about.pH 7.2)
[0329] 150 .mu.M sHh-N conjugated to N-octylmaleimide
[0330] 150 .mu.M DTT conjugated to N-octylmaleimide
[0331] 500 .mu.M DTT
[0332] 0.5% DMSO
[0333] Approximate Composition of the N-octylmaleimide Vehicle
Solution
[0334] PBS (.about.pH 7.2)
[0335] 150 .mu.M DTT conjugated to N-octylmaleimide
[0336] 500 .mu.M DTT
[0337] 0.5% DMSO
[0338] 0.5% DMSO
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[0427] Equivalents
[0428] While we have described a number of embodiments of this
invention, it is apparent to persons having ordinary skill in the
art that our basic embodiments may be altered to provide other
embodiments that utilize the compositions and processes of this
invention. Therefore, it will be appreciated that the scope of this
invention includes all alternative embodiments and variations which
are defined in the foregoing specificafion and by the claims
appended hereto; and the invention is not to be limited by the
specific embodiments presented in the examples.
Sequence CWU 1
1
4 1 175 PRT human 1 Cys Gly Pro Gly Arg Val Val Gly Ser Arg Arg Arg
Pro Pro Arg Lys 1 5 10 15 Leu Val Pro Leu Ala Tyr Lys Gln Phe Ser
Pro Asn Val Pro Glu Lys 20 25 30 Thr Leu Gly Ala Ser Gly Arg Tyr
Glu Gly Lys Ile Ala Arg Ser Ser 35 40 45 Glu Arg Phe Lys Glu Leu
Thr Pro Asn Tyr Asn Pro Asp Ile Ile Phe 50 55 60 Lys Asp Glu Glu
Asn Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cys 65 70 75 80 Lys Asp
Arg Leu Asn Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pro 85 90 95
Gly Val Lys Leu Arg Val Thr Glu Gly Trp Asp Glu Asp Gly His His 100
105 110 Ser Glu Glu Ser Leu His Tyr Glu Gly Arg Ala Val Asp Ile Thr
Thr 115 120 125 Ser Asp Arg Asp Arg Asn Lys Tyr Gly Leu Leu Ala Arg
Leu Ala Val 130 135 140 Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu Ser
Lys Ala His Val His 145 150 155 160 Cys Ser Val Lys Ser Glu His Ser
Ala Ala Ala Lys Thr Gly Gly 165 170 175 2 174 PRT human 2 Cys Gly
Pro Gly Arg Gly Phe Gly Lys Arg Arg His Pro Lys Lys Leu 1 5 10 15
Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro Asn Val Ala Glu Lys Thr 20
25 30 Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys Ile Ser Arg Asn Ser
Glu 35 40 45 Arg Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pro Asp Ile
Ile Phe Lys 50 55 60 Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu Met
Thr Gln Arg Cys Lys 65 70 75 80 Asp Lys Leu Asn Ala Leu Ala Ile Ser
Val Met Asn Gln Trp Pro Gly 85 90 95 Val Lys Leu Arg Val Thr Glu
Gly Trp Asp Glu Asp Gly His His Ser 100 105 110 Glu Glu Ser Leu His
Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser 115 120 125 Asp Arg Asp
Arg Ser Lys Tyr Gly Met Leu Ala Arg Leu Ala Val Glu 130 135 140 Ala
Gly Phe Asp Trp Val Tyr Tyr Glu Ser Lys Ala His Ile His Cys 145 150
155 160 Ser Val Lys Ala Glu Asn Ser Val Ala Ala Lys Ser Gly Gly 165
170 3 176 PRT human 3 Cys Gly Pro Gly Arg Gly Pro Val Gly Arg Arg
Arg Tyr Ala Arg Lys 1 5 10 15 Gln Leu Val Pro Leu Leu Tyr Lys Gln
Phe Val Pro Gly Val Pro Glu 20 25 30 Arg Thr Leu Gly Ala Ser Gly
Pro Ala Glu Gly Arg Val Ala Arg Gly 35 40 45 Ser Glu Arg Phe Arg
Asp Leu Val Pro Asn Tyr Asn Pro Asp Ile Ile 50 55 60 Phe Lys Asp
Glu Glu Asn Ser Gly Ala Asp Arg Leu Met Thr Glu Arg 65 70 75 80 Cys
Lys Glu Arg Val Asn Ala Leu Ala Ile Ala Val Met Asn Met Trp 85 90
95 Pro Gly Val Arg Leu Arg Val Thr Glu Gly Trp Asp Glu Asp Gly His
100 105 110 His Ala Gln Asp Ser Leu His Tyr Glu Gly Arg Ala Leu Asp
Ile Thr 115 120 125 Thr Ser Asp Arg Asp Arg Asn Lys Tyr Gly Leu Leu
Ala Arg Leu Ala 130 135 140 Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr
Glu Ser Arg Asn His Val 145 150 155 160 His Val Ser Val Lys Ala Asp
Asn Ser Leu Ala Val Arg Ala Gly Gly 165 170 175 4 176 PRT human
SITE (6) Xaa= Val or Gly 4 Cys Gly Pro Gly Arg Xaa Xaa Xaa Xaa Xaa
Arg Arg Xaa Xaa Xaa Lys 1 5 10 15 Xaa Leu Xaa Pro Leu Xaa Tyr Lys
Gln Phe Xaa Pro Xaa Val Xaa Glu 20 25 30 Lys Thr Leu Gly Ala Ser
Gly Arg Xaa Glu Gly Lys Xaa Xaa Arg Xaa 35 40 45 Ser Glu Arg Phe
Lys Xaa Leu Xaa Pro Asn Tyr Asn Pro Asp Ile Ile 50 55 60 Phe Lys
Asp Glu Glu Asn Xaa Gly Ala Asp Arg Leu Met Thr Xaa Arg 65 70 75 80
Cys Lys Xaa Xaa Xaa Asn Ser Leu Ala Ile Xaa Val Met Asn Xaa Trp 85
90 95 Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp Asp Glu Asp Gly
His 100 105 110 His Xaa Xaa Xaa Ser Leu His Tyr Glu Gly Arg Ala Val
Asp Ile Thr 115 120 125 Thr Ser Asp Arg Asp Arg Xaa Lys Tyr Gly Xaa
Leu Ala Arg Leu Ala 130 135 140 Val Glu Ala Gly Phe Asp Trp Val Tyr
Tyr Glu Ser Xaa Xaa His Xaa 145 150 155 160 His Xaa Ser Val Lys Xaa
Xaa Xaa Ser Xaa Ala Ala Xaa Xaa Gly Gly 165 170 175
* * * * *