U.S. patent application number 13/062297 was filed with the patent office on 2011-06-30 for isolation and identification of glycosaminoglycans.
This patent application is currently assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH. Invention is credited to Simon McKenzie Cool, Christian Dombrowski, Victor Nurcombe.
Application Number | 20110159071 13/062297 |
Document ID | / |
Family ID | 40042333 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110159071 |
Kind Code |
A1 |
Cool; Simon McKenzie ; et
al. |
June 30, 2011 |
Isolation and Identification of Glycosaminoglycans
Abstract
The isolation and identification of glycosaminoglycans capable
of binding to proteins having a heparin-binding domain is
disclosed, as well as the use of the glycosaminoglycans isolated in
the growth and/or development of tissue.
Inventors: |
Cool; Simon McKenzie;
(Singapore, SG) ; Nurcombe; Victor; (Singapore,
SG) ; Dombrowski; Christian; (Singapore, SG) |
Assignee: |
AGENCY FOR SCIENCE, TECHNOLOGY AND
RESEARCH
Singapore
SG
|
Family ID: |
40042333 |
Appl. No.: |
13/062297 |
Filed: |
February 19, 2009 |
PCT Filed: |
February 19, 2009 |
PCT NO: |
PCT/GB09/00469 |
371 Date: |
March 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61096274 |
Sep 11, 2008 |
|
|
|
Current U.S.
Class: |
424/423 ;
424/93.7; 435/325; 435/383; 435/7.1; 514/16.5; 514/16.7; 514/17.1;
514/20.9; 530/322; 536/123.1 |
Current CPC
Class: |
A61K 31/727 20130101;
B01D 15/3823 20130101; A61L 27/365 20130101; A61L 2300/414
20130101; A61K 31/737 20130101; A61L 2430/02 20130101; A61L
2300/236 20130101; A61K 38/1875 20130101; A61P 19/08 20180101; A61P
43/00 20180101; C07K 14/51 20130101; A61K 35/28 20130101; A61L
27/3847 20130101; A61K 2035/124 20130101; C12N 2501/155 20130101;
C12N 5/0663 20130101; A61L 2300/252 20130101; A61P 17/02 20180101;
C12N 2501/90 20130101; A61P 19/00 20180101; A61L 27/26 20130101;
C08B 37/0003 20130101; A61L 27/54 20130101; A61L 27/3834 20130101;
C08B 37/0075 20130101 |
Class at
Publication: |
424/423 ;
424/93.7; 435/7.1; 435/325; 435/383; 514/16.5; 514/16.7; 514/17.1;
514/20.9; 530/322; 536/123.1 |
International
Class: |
A61F 2/00 20060101
A61F002/00; A61K 35/12 20060101 A61K035/12; G01N 33/53 20060101
G01N033/53; C12N 5/077 20100101 C12N005/077; C12N 5/02 20060101
C12N005/02; A61P 17/02 20060101 A61P017/02; A61P 19/00 20060101
A61P019/00; A61P 43/00 20060101 A61P043/00; A61K 38/14 20060101
A61K038/14; C07K 9/00 20060101 C07K009/00; C07H 1/00 20060101
C07H001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 6, 2008 |
GB |
0818255.2 |
Claims
1. A method of isolating glycosaminoglycans capable of binding to a
protein having a heparin-binding domain, the method comprising: (i)
providing a solid support having polypeptide molecules adhered to
the support, wherein the polypeptide comprises a heparin-binding
domain; (ii) contacting the polypeptide molecules with a mixture
comprising glycosaminoglycans such that
polypeptide-glycosaminoglycan complexes are allowed to form; (iii)
partitioning polypeptide-glycosaminoglycan complexes from the
remainder of the mixture; (iv) dissociating glycosaminoglycans from
the polypeptide-glycosaminoglycan complexes; (v) collecting the
dissociated glycosaminoglycans.
2. A method of identifying glycosaminoglycans capable of
stimulating or inhibiting the growth and/or differentiation of
cells and/or tissues, the method comprising: (i) providing a solid
support having polypeptide molecules adhered to the support,
wherein the polypeptide comprises a heparin-binding domain; (ii)
contacting the polypeptide molecules with a mixture comprising
glycosaminoglycans such that polypeptide-glycosaminoglycan
complexes are allowed to form; (iii) partitioning
polypeptide-glycosaminoglycan complexes from the remainder of the
mixture; (iv) dissociating glycosaminoglycans from the
polypeptide-glycosaminoglycan complexes; (v) collecting the
dissociated glycosaminoglycans; (vi) adding the collected
glycosaminoglycans to cells or tissues in which a protein
containing the amino acid sequence of the heparin-binding domain is
present; (vii) measuring one or more of: proliferation of the
cells, differentiation of the cells, expression of one or more
protein markers.
3. The method of claim 1 or 2 wherein the mixture comprising
glycosaminoglycans contains extracellular matrix material.
4. The method of claim 3 wherein the extracellular matrix material
is derived from connective tissue or connective tissue cells.
5. The method of any one of claims 1 to 4 wherein the mixture
comprising glycosaminoglycans contains one or more of a dextran
sulphate, a chondroitin sulphate, a heparan sulphate.
6. The method of any one of claims 1 to 5 wherein the mixture
comprising glycosaminoglycans has been enriched for one of dextran
sulphate, chondroitin sulphate, heparan sulphate.
7. The method of any one of claims 1 to 6 wherein the method
further comprises subjecting the collected glycosaminoglycans to
further analysis in order to determine structural characteristics
of the GAG.
8. The method of any one of claims 1 to 7 wherein the
glycosaminoglycan-polypeptide complexes are contacted with a
lyase.
9. The method of any one of claims 1 to 8 wherein the polypeptide
is, or comprises, one of SEQ ID NO.s: 1-13 or 17.
10. Heparan sulphate HS/BMP2.
11. Culture media comprising HS/BMP2.
12. A pharmaceutical composition or medicament comprising
HS/BMP2.
13. The pharmaceutical composition or medicament of claim 12
further comprising a pharmaceutically acceptable carrier, adjuvant
or diluent.
14. The pharmaceutical composition or medicament of claim 12 or 13
further comprising BMP2 protein.
15. A pharmaceutical compositions or medicament according to any
one of claims 12 to 14 for use in the prevention or treatment of
injury or disease.
16. Use of HS/BMP2 in the manufacture of a medicament for the
prevention or treatment of injury or disease.
17. The pharmaceutical composition or use of claim 15 or 16 wherein
the prevention or treatment is chosen from: repair, regeneration or
replacement of connective tissue and wound healing.
18. A method of preventing or treating injury or disease in a
patient in need of treatment thereof, the method comprising
administering an effective amount of HS/BMP2 to the patient.
19. The method of claim 18 wherein the administered HS/BMP2 is
formulated as a pharmaceutical composition or medicament.
20. The method of claim 19 wherein the pharmaceutical composition
or medicament further comprises BMP2 protein.
21. A method of promoting or inhibiting osteogenesis comprising
administering HS/BMP2 to bone precursor cells or bone stem
cells.
22. The method of claim 21 wherein the bone precursor cells or bone
stem cells are contacted with HS/BMP2 in vitro.
23. The method of claim 21 wherein the bone precursor cells or bone
stem cells are contacted with HS/BMP2 in vivo.
24. The method of any one of claims 21 to 23 wherein the bone
precursor cells or bone stem cells are contacted with BMP2
simultaneously with HS/BMP2.
25. A method of promoting or inhibiting the formation of cartilage
tissue comprising administering HS/BMP2 to cartilage precursor
cells or cartilage stem cells.
26. The method of claim 25 wherein the cartilage precursor cells or
cartilage stem cells are contacted with HS/BMP2 in vitro.
27. The method of claim 25 wherein the cartilage precursor cells or
cartilage stem cells are contacted with HS/BMP2 in vivo.
28. The method of any one of claims 25 to 27 wherein the cartilage
precursor cells or cartilage stem cells are contacted with BMP2
simultaneously with HS/BMP2.
29. A method for the repair, replacement or regeneration of
connective tissue in a human or animal patient in need of such
treatment, the method comprising: (i) culturing mesenchymal stem
cells in vitro in contact with HS/BMP2 for a period of time
sufficient for said cells to form connective tissue; (ii)
collecting said connective tissue; (iii) implanting said connective
tissue into the body of the patient at a site of injury or disease
to repair, replace or regenerate connective tissue in the
patient.
30. The method of claim 29 further comprising contacting the
mesenchymal cells in culture with exogenous BMP2.
31. Connective tissue obtained by in vitro culture of mesenchymal
stem cells in the presence of HS/BMP2.
32. Connective tissue as claimed in claim 31, wherein the
mesenchymal cells are cultured in the presence of exogenous BMP2,
and optionally in the presence of BMP2.
33. A pharmaceutical composition comprising stem cells and
HS/BMP2.
34. The pharmaceutical composition of claim 32 wherein the stem
cells are mesenchymal stem cells.
35. The pharmaceutical composition of claim 33 or 34 wherein the
composition further comprises BMP2.
36. A pharmaceutical composition according to any of claims 33 to
35 for use in the treatment of injury or disease.
37. A method for the treatment of injury or disease in a patient in
need of treatment thereof comprising administering to the patient a
pharmaceutical composition comprising stem cells and HS/BMP2.
38. The method of claim 37 wherein the stem cells are mesenchymal
stem cells.
39. The method of claim 37 or 38 wherein the method further
comprises administering BMP2 to the patient.
40. Use of HS/BMP2 in the growth of connective tissue in vitro.
41. A method for growing connective tissue in vitro comprising
culturing mesenchymal stem cells in contact with exogenously added
HS/BMP2.
42. A biological implant comprising a solid or semi-solid matrix
material impregnated with HS/BMP2.
43. The biological implant of claim 42 further impregnated with
BMP2.
44. The biological implant of claim 42 or 43 further impregnated
with mesenchymal stem cells.
45. A kit comprising a predetermined amount of a glycosaminoglycan
having high affinity for a protein having a heparin-binding domain
and a predetermined amount of said protein.
46. The kit of claim 45 wherein the glycosaminoglycan is HS/BMP2
and the protein is BMP2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the isolation and
identification of glycosaminoglycans capable of binding to proteins
having a heparin-binding domain, as well as to the use of the
glycosaminoglycans isolated in the growth and/or development of
tissue.
BACKGROUND TO THE INVENTION
[0002] Glycosaminoglycans (GAGs) are complex carbohydrate
macromolecules responsible for performing and regulating a vast
number of essential cellular functions.
[0003] GAGs have been implicated in the modulation or mediation of
many signalling systems in concert with the many hundreds of known
heparin-binding growth and adhesive factors. It is contemplated
that the association of growth factors with GAGs modulates their
various activities with a diverse range of actions, such as
lengthening their half-lives by protecting them from proteolytic
degradation, modulating localisation of these cytokines at the cell
surface, mediating molecular interactions and stabilising
ligand-receptor complexes.
[0004] There are an ever increasing number of identified
heparin-binding growth factors, adding to the hundreds already
known, most of which were purified by heparin affinity
chromatography. They include the extensive fibroblast growth factor
(FGF) family, the PDGFs and the pleiotropins through to the
TGF-.beta. superfamily of cytokines. This latter family of factors
encompasses the osteo-inductive bone morphogenetic protein (BMP)
subfamily, so named for their ability to induce ectopic bone
formation.
[0005] The nature and effect of the interaction of GAGs and growth
factors remains unclear. Although the interaction between FGF2 and
particular saccharide sequences found within heparin has been shown
to be of high affinity, it remains generally unclear whether the
association between other growth factors and heparans involves a
high affinity or specific binding interaction between an amino acid
sequence epitope on the protein growth factor and a saccharide
sequence embedded in the GAG, or whether the association is
mediated by lower affinity, non-specific interactions between the
GAG and protein growth factor.
[0006] If interactions between GAGs and proteins resident in, or
secreted into, the extracellular matrix are specific, the binding
partners need to be identified in order to unravel the interactions
and understand how these interactions may be used or modulated to
provide new treatments.
[0007] A major question that arises is, therefore, whether there
are saccharide sequences embedded in the chains of GAG molecules
that match primary amino acid sequences within the polypeptide
backbone of growth factors so controlling their association, and so
bioactivity, with absolute, or at least relative, specificity.
SUMMARY OF THE INVENTION
[0008] We have devised a method to answer this question which
involves enriching for glycosaminoglycan molecules that exhibit
binding to particular polypeptides having a heparin-binding domain.
Isolated GAG mixtures and/or molecules can then be identified and
tested for their ability to modulate the growth and differentiation
of cells and tissue expressing a protein containing the
heparin-binding domain. For the first time, this enables the
controlled analysis of the effect of particular GAG saccharide
sequences on the growth and differentiation of cells and tissue,
both in vitro and in vivo.
[0009] Accordingly, in a first aspect of the present invention a
method of isolating glycosaminoglycans capable of binding to
proteins having heparin/heparan-binding domains is provided, the
method comprising: [0010] (i) providing a solid support having
polypeptide molecules adhered to the support, wherein the
polypeptide comprises a heparin-binding domain; [0011] (ii)
contacting the polypeptide molecules with a mixture comprising
glycosaminoglycans such that polypeptide-glycosaminoglycan
complexes are allowed to form; [0012] (iii) partitioning
polypeptide-glycosaminoglycan complexes from the remainder of the
mixture; [0013] (iv) dissociating glycosaminoglycans from the
polypeptide-glycosaminoglycan complexes; [0014] (v) collecting the
dissociated glycosaminoglycans.
[0015] In another aspect of the present invention isolated
glycosaminoglycans are identified by their ability to modulate the
growth or differentiation of cells or tissues. A method of
identifying glycosaminoglycans capable of stimulating or inhibiting
the growth and/or differentiation of cells and/or tissues is
provided, the method comprising: [0016] (i) providing a solid
support having polypeptide molecules adhered to the support,
wherein the polypeptide comprises a heparin-binding domain; [0017]
(ii) contacting the polypeptide molecules with a mixture comprising
glycosaminoglycans such that polypeptide-glycosaminoglycan
complexes are allowed to form; [0018] (iii) partitioning
polypeptide-glycosaminoglycan complexes from the remainder of the
mixture; [0019] (iv) dissociating glycosaminoglycans from the
polypeptide-glycosaminoglycan complexes; [0020] (v) collecting the
dissociated glycosaminoglycans; [0021] (vi) adding the collected
glycosaminoglycans to cells or tissues in which a protein
containing the amino acid sequence of the heparin-binding domain is
present; [0022] (vii) measuring one or more of: proliferation of
the cells, differentiation of the cells, expression of one or more
protein markers.
[0023] In embodiments of the present invention the mixture
comprising GAGs may contain synthetic glycosaminoglycans. However,
in preferred embodiments GAGs obtained from cells or tissues are
used. For example, the mixture may contain extracellular matrix
wherein the extracellular matrix material is obtained by scraping
live tissue in situ (i.e. directly from the tissue in the body of
the human or animal from which it is obtained) or by scraping
tissue (live or dead) that has been extracted from the body of the
human or animal. Alternatively, the extracellular matrix material
may be obtained from cells grown in culture. The extracellular
matrix material may be obtained from connective tissue or
connective tissue cells, e.g. bone, cartilage, muscle, fat,
ligament or tendon.
[0024] The GAG component may be extracted from a tissue or cell
sample or extract by a series of routine separation steps (e.g.
anion exchange chromatography), well known to those of skill in the
art.
[0025] GAG mixtures may contain a mixture of different types of
glycosaminoglycan, which may include dextran sulphates, chondroitin
sulphates and heparan sulphates. In preferred embodiments the GAG
mixture contacted with the solid support has been enriched for one
of these types of glycosaminoglycan, most preferably for heparan
sulphate. A heparan sulphate-, chondroitin sulphate- or dextran
sulphate-enriched GAG fraction may be obtained by performing column
chromatography on the GAG mixture, e.g. weak, medium or strong
anion exchange chromatography, as well as strong high pressure
liquid chromatography (SAX-HPLC), with selection of the appropriate
fraction.
[0026] The collected GAGs may be subjected to further analysis in
order to identify the GAG, e.g. determine GAG composition or
sequence, or determine structural characteristics of the GAG. GAG
structure is typically highly complex, and, taking account of
currently available analytical techniques, exact determinations of
GAG sequence structure are not possible in most cases.
[0027] However, the collected GAG molecules may be subjected to
partial or complete saccharide digestion (e.g. chemically by
nitrous acid or enzymatically with lyases such as heparinase III)
to yield saccharide fragments that are both characteristic and
diagnostic of the GAG. In particular, digestion to yield
disaccharides (or tetrasaccharides) may be used to measure the
percentage of each disaccharide obtained which will provide a
characteristic disaccharide "fingerprint" of the GAG.
[0028] The pattern of sulphation of the GAG can also be determined
and used to determine GAG structure. For example, for heparan
sulphate the pattern of sulphation at amino sugars and at the C2,
C3 and C6 positions may be used to characterise the heparan
sulphate.
[0029] Disaccharide analysis, tetrasaccharide analysis and analysis
of suphation can be used in conjunction with other analytical
techniques such as HPLC, mass spectrometry and NMR which can each
provide unique spectra for the GAG. In combination, these
techniques may provide a definitive structural characterisation of
the GAG.
[0030] A high affinity binding interaction between the GAG and
heparin-binding domain indicates that the GAG will contain a
specific saccharide sequence that contributes to the high affinity
binding interaction. A further step may comprise determination of
the complete or partial saccharide sequence of the GAG, or the key
portion of the GAG, involved in the binding interaction.
[0031] In one embodiment, GAG-polypeptide complexes may be
subjected to treatment with an agent that lyses glycosaminoglycan
chains, e.g. a lyase. Lyase treatment may cleave portions of the
bound GAG that are not taking part in the binding interaction with
the polypeptide. Portions of the GAG that are taking part in the
binding interaction with the polypeptide may be protected from
lyase action. After removal of the lyase, e.g. following a washing
step, the GAG molecule that remains bound to the polypeptide
represents the specific binding partner ("GAG ligand") of the
polypeptide. Owing to the lower complexity of shorter GAG
molecules, following dissociation and collection of the GAG ligand,
a higher degree of structural characterisation of the GAG ligand
can be expected. For example, the combination of any of the
saccharide sequence (i.e. the primary (linear) sequence of
monosaccharides contained in the GAG ligand), sulphation pattern,
disaccharide and/or tetrasaccharide digestion analysis, NMR
spectra, mass spectrometry spectra and HPLC spectra may provide a
high level of structural characterisation of the GAG ligand.
[0032] In one aspect of the present invention a GAG is provided
having high binding affinity for BMP2. More preferably the GAG is a
heparan sulphate (HS). The HS was isolated from a GAG mixture
obtained from the extracellular matrix of osteoblasts by following
the methodology of the present invention in which a polypeptide
comprising the heparin-binding domain of BMP2 (SEQ ID NO:1) was
attached to a solid support and GAG-polypeptide complexes were
allowed to form. Dissociation of the GAG component from the
GAG-polypeptide complexes led to isolation of a unique HS herein
called "HS/BMP2".
[0033] Accordingly, in one aspect of the present invention HS/BMP2
is provided. HS/BMP2 may be provided in isolated or purified form.
In another aspect culture media comprising HS/BMP2 is provided.
[0034] In yet another aspect of the present invention a
pharmaceutical composition or medicament comprising HS/BMP2 is
provided, optionally in combination with a pharmaceutically
acceptable carrier, adjuvant or diluent. In some embodiments
pharmaceutical compositions or medicaments may further comprise
BMP2 protein. Pharmaceutical compositions or medicaments comprising
HS/BMP2 are provided for use in the prevention or treatment of
injury or disease. The use of HS/BMP2 in the manufacture of a
medicament for the prevention or treatment of injury or disease is
also provided.
[0035] In a further aspect of the present invention, a method of
preventing or treating injury or disease in a patient in need of
treatment thereof is provided, the method comprising administering
an effective amount of HS/BMP2 to the patient. The administered
HS/BMP2 may be formulated in a suitable pharmaceutical composition
or medicament and may further comprise a pharmaceutically
acceptable carrier, adjuvant or diluent. Optionally, the
pharmaceutical composition or medicament may also comprise BMP2
protein.
[0036] In another aspect of the present invention a method of
promoting or inhibiting osteogenesis (the formation of bone cells
and/or bone tissue) is provided comprising administering HS/BMP2 to
bone precursor cells or bone stem cells.
[0037] In another aspect of the present invention a method of
promoting or inhibiting the formation of cartilage tissue
(chondrogenesis) is provided, comprising administering HS/BMP2 to
cartilage precursor cells or cartilage stem cells.
[0038] The methods of stimulating or inhibiting osteogenesis or
formation of cartilage tissue may be conducted in vitro by
contacting bone or cartilage precursor or stem cells with HS/BMP2,
optionally in the presence of exogenously added BMP2 protein. The
precursor cells or stem cells may be mesenchymal stem cells. Where
tissue formation is promoted, the tissue formed may be collected
and used for implantation into a human or animal patient.
[0039] Accordingly, in one aspect of the present invention,
connective tissue is provided wherein the connective tissue is
obtained by in vitro culture of mesenchymal stem cells in the
presence of HS/BMP2 (i.e. exogenous HS/BMP2), and optionally in the
presence of BMP2 (i.e. exogenous BMP2). The connective tissue may
be bone, cartilage, muscle, fat, ligament or tendon.
[0040] The prevention or treatment of disease using HS/BMP2 may
involve the repair, regeneration or replacement of tissue,
particularly connective tissue such as bone, cartilage, muscle,
fat, ligament or tendon.
[0041] In patients having a deterioration of one of these tissues,
administration of HS/BMP2 to the site of deterioration may be used
to stimulate the growth, proliferation and/or differentiation of
tissue at that site. For example, stimulation of mesenchymal stem
cells present at, or near to, the site of administration may lead,
preferably when BMP2 is also present at the site, to the
proliferation and differentiation of the mesenchymal stem cells
into the appropriate connective tissue, thereby providing for
replacement/regeneration of the damaged tissue and treatment of the
injury.
[0042] Alternatively, connective tissue obtained from in vitro
culture of mesenchymal stem cells in contact with HS/BMP2 may be
collected and implanted at the site of injury or disease to replace
damaged or deteriorated tissue. The damaged or deteriorated tissue
may optionally first be excised from the site of injury or
disease.
[0043] In another aspect, a pharmaceutical composition may be
provided containing stem cells, preferably mesenchymal stem cells,
and HS/BMP2. Administration, e.g. injection, of the composition at
the site of injury, disease or deterioration provides for the
regeneration of tissue at the site.
[0044] Accordingly, HS/BMP2 is useful in wound healing in vivo,
including tissue repair, regeneration and/or replacement (e.g.
healing of scar tissue or a broken bone) effected by direct
application of HS/BMP2, optionally in combination with BMP2 and/or
stem cells, to the patient requiring treatment. HS/BMP2 is also
useful in the in vitro generation of tissue suitable for
implantation into a patient in need of tissue repair, regeneration
and/or replacement.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0045] The present invention relates to GAGs, and especially to
methods of enriching mixtures of compounds containing one or more
GAGs that bind to a polypeptide corresponding to the
heparin-binding domain of a protein that binds heparin/heparan (a
"heparin-binding factor"). Enrichment leads to isolation of GAGs,
whether as a mixture containing different GAGs or a population of
GAGs that are structurally or functionally identical (or
substantially identical). The enriched mixture preferably has a
modulating effect on the heparin-binding factor.
[0046] The present invention also relates to mixtures of compounds
enriched with one or more GAGs which possess a modulating effect on
a heparin/heparan-binding factor, and methods of using such
mixtures.
[0047] The present invention also relates to GAG molecules which
potentiate (e.g. agonize) the activity of BMP-2 and hence its
ability to stimulate stem cell proliferation and bone
formation.
[0048] As used herein, the terms `enriching`, `enrichment`,
`enriched`, etc. describes a process (or state) whereby the
relative composition of a mixture is (or has been) altered in such
a way that the fraction of that mixture given by one or more of
those entities is increased, while the fraction of that mixture
given by one or more different entities is decreased.
[0049] GAGs isolated by enrichment may be pure, i.e. contain
substantially only one type of GAG, or may continue to be a mixture
of different types of GAG, the mixture having a higher proportion
of particular GAGs that bind to the heparin-binding domain relative
to the starting mixture.
[0050] GAGs identified by the present invention are preferably GAGs
that exhibit a functional effect when contacted with cells or
tissue in which a protein containing the heparin-binding domain is
expressed or contained. The functional effect may be a modulating
or potentiating effect.
[0051] The functional effect may be to promote (stimulate) or
inhibit the proliferation of the cells of a certain type or the
differentiation of one cell type into another, or the expression of
one or more protein markers. For example, the GAGs may promote cell
proliferation, i.e. an increase in cell number, or promote
differentiation of stem cells into specialised cell types (e.g.
mesenchymal stem cells in connective tissue), promote or inhibit
the expression of protein markers indicative of the multipotency or
differentiation state of the cells (e.g. markers such as alkaline
phosphatase activity, detection of RUNX2, osterix, collagen I, II,
IV, VII, X, osteopontin, Osteocalcin, BSPII, SOX9, Aggrecan, ALBP,
CCAAT/enhancer binding protein-.alpha. (C/EBP.alpha.), adipocyte
lipid binding protein (ALBP), alkaline phosphatise (ALP), bone
sialoprotein 2, (BSPII), Collagen2a1 (CoII2a) and SOX9).
[0052] As used herein, the term `modulating effect` is understood
to mean the effect that a first entity has on a second entity
wherein the second entity's normal function in another process or
processes is modified by the presence of the first entity. In a
preferred embodiment of the present invention, the modulating
effect may be either agonistic or antagonistic.
[0053] The modulating effect may be a potentiating effect. The term
`potentiating effect` is understood to mean the effect of
increasing potency. In a preferred embodiment of the present
invention, the term `potentiating effect` refers to the effect that
a first entity has on a second entity, which effect increases the
potency of that second entity in another process or processes. In a
further preferred embodiment of the present invention, the
potentiating effect is understood to mean the effect of isolated
GAGs on a heparin-binding factor, wherein the said effect increases
the potency of said heparin-binding factor.
[0054] In a preferred embodiment of the present invention, the
potentiating effect is an increase in bioavailability of the
heparin-binding factor. In a preferred embodiment of the present
invention, the potentiating effect is an increase in
bioavailability of BMP2. One method of measuring an increase in
bioavailability of the heparin-binding factor is through
determining an increase in local concentration of the
heparin-binding factor.
[0055] In another embodiment of the present invention, the
potentiating effect is to protect the heparin-binding factor from
degradation. In an especially preferred embodiment of the present
invention, the potentiating effect is to protect BMP-2 from
degradation. One method of determining a decrease in the
degradation of the heparin-binding factor is through measuring an
increase in the half-life of the heparin-binding factor.
[0056] In another embodiment of the present invention, the
potentiating effect is to sequester heparin-binding factors away
from cellular receptors. In another embodiment of the present
invention, the potentiating effect is to stabilise the
ligand-receptor interaction.
[0057] The potentiating effect (e.g. modulation of growth or
differentiation) may be determined by use of appropriate assays.
For example, the effect that an HS has on the stability of BMP-2
may be determined by ELISA. The effect that an HS has on the
activity of BMP-2 may be determined by measuring the
activation/expression of one or more of SMAD 1, 5 or 8, or
measuring the expression of one or more osteogenic marker genes
such as Runx2, alkaline phosphatase, Osterix, Osteocalcin and BSP1,
or measuring the levels of mineralization using staining such as
Alizarin Red and von Kossa.
[0058] As used herein, the process of `contacting` involves the
bringing into close physical proximity of two or more discrete
entities. The process of `contacting` involves the bringing into
close proximity of two or more discrete entities for a time, and
under conditions, sufficient to allow a portion of those two or
more discrete entities to interact on a molecular level.
Preferably, as used herein, the process of `contacting` involves
the bringing into close proximity of the mixture of compounds
possessing one or more GAGs and the polypeptide corresponding to
the heparin-binding domain of a heparin-binding factor. Examples of
`contacting` processes include mixing, dissolving, swelling,
washing. In preferred embodiments `contact` of the GAG mixture and
polypeptide is sufficient for complexes, which may be covalent but
are preferably non-covalent, to form between GAGs and polypeptides
that exhibit high affinity for each other.
[0059] The polypeptide may comprise the full length or near full
length primary amino acid sequence of a selected protein having a
heparin-binding domain. Due to folding that may occur in longer
polypeptides leading to possible masking of the heparin-binding
domain from the GAG mixture, it is preferred for the polypeptide to
be short. Preferably, the polypeptide will have an amino acid
sequence that includes the heparin-binding domain and optionally
including one or more amino acids at one or each of the N- and
C-terminals of the peptides. These additional peptides may enable
the addition of linker or attachment molecules to the polypeptide
that are required to attach the polypeptide to the solid
support.
[0060] In preferred embodiments in addition to the number of amino
acids in the heparin-binding domain the polypeptide contains 1-20,
more preferably 1-10, still more preferably 1-5 additional amino
acids. In some embodiments the amino acid sequence of the
heparin-binding domain accounts for at least 80% of the amino acids
of the polypeptide, more preferably at least 90%, still more
preferably at least 95%.
[0061] In order to adhere polypeptides to the surface of a solid
support the polypeptides are preferably modified to include a
molecular tag, and the surface of the solid support is modified to
incorporate a corresponding molecular probe having high affinity
for the molecular tag, i.e. the molecular tag and probe form a
binding pair. In preferred embodiments the tag and/or probe is
chosen from any one of: an antibody, a cell receptor, a ligand,
biotin, any fragment or derivative of these structures, any
combination of the foregoing, or any other structure with which a
probe can be designed or configured to bind or otherwise associate
with specificity. A preferred binding pair suitable for use as tag
and probe is biotin and avidin.
[0062] The polypeptide is preferably derived from a protein of
interest. By "derived from" is meant that the polypeptide is
chosen, selected or prepared because it contains the amino acid
sequence of a heparin-binding domain that is present in a protein
of interest. In some embodiments, the amino acid sequence of the
heparin-binding domain may be modified from that appearing in the
protein of interest, e.g. to investigate the effect of changes in
the heparin-binding domain sequence on GAG binding.
[0063] The protein of interest may be any protein that binds
heparin, and therefore has a heparin-binding domain. Preferred
proteins include those expressed in the extracellular matrix, in
particular in the extracellular matrix of connective tissue (e.g.
bone, cartilage, muscle, tendons, ligaments, fat).
[0064] Preferred proteins and their heparin-binding domains are set
out below:
TABLE-US-00001 SEQ Amino acid sequence of ID Protein
heparin-binding domain NO. BMP2 QAKHKQRKRLKSSCKRHP 1 BMP4
SPKHHSQRARKKNKNCRRH 2 FGF2 TYRSRKYTSWYVALKRTGQYKLGSKTGPGQK 3 SHH
GKRRHPKKLTPLAYKQ 4 VEGF KCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWS 5
189 FGFR1 APYWTSPEKMEKKLHAVPAAKTVK 6 VITRO-
RPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRR 7 NECTIN PDGF B
RVRRPPKGKHRKFKHTH 8 HB-EGF HGKRKKKGKGLGKKRDPCLRKYK 9 FGFR3
APYWTRPERMDKKLLAVPAANTVR 10 FIBRO- TLENVSPPRRARV 11 NECTIN LAMININ
RYVVLPRPVCFEKGMNYTVR 12 N-CAM IWKHKGRDVILKKDVRFI 13
[0065] It is understood by those skilled in the art that small
variations in the amino acid sequence of a particular polypeptide
may allow the inherent functionality of that portion to be
maintained. It is also understood that the substitution of certain
amino acid residues within a peptide with other amino acid residues
that are isosteric and/or isoelectronic may either maintain or
improve certain properties of the unsubstituted peptide. These
variations are also encompassed within the scope of the present
invention. For example, the amino acid alanine may sometimes be
substituted for the amino acid glycine (and vice versa) whilst
maintaining one or more of the properties of the peptide. The term
`isosteric` refers to a spatial similarity between two entities.
Two examples of moieties that are isosteric at moderately elevated
temperatures are the iso-propyl and tert-butyl groups. The term
`isoelectronic` refers to an electronic similarity between two
entities, an example being the case where two entities possess a
functionality of the same, or similar, pKa.
[0066] In embodiments of the present invention, the polypeptide
corresponding to the heparin-binding domain may be synthetic or
recombinant.
[0067] The solid support may be any substrate having a surface to
which molecules may be attached, directly or indirectly, through
either covalent or non-covalent bonds. The solid support may
include any substrate material that is capable of providing
physical support for the probes that are attached to the surface.
It may be a matrix support. The material is generally capable of
enduring conditions related to the attachment of the probes to the
surface and any subsequent treatment, handling, or processing
encountered during the performance of an assay. The materials may
be naturally occurring, synthetic, or a modification of a naturally
occurring material. The solid support may be a plastics material
(including polymers such as, e.g., poly(vinyl chloride),
cyclo-olefin copolymers, polyacrylamide, polyacrylate,
polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polymethacrylate, poly(ethylene terephthalate),
polytetrafluoroethylene (PTFE or Teflon.RTM.), nylon, poly(vinyl
butyrate)), etc., either used by themselves or in conjunction with
other materials. Additional rigid materials may be considered, such
as glass, which includes silica and further includes, for example,
glass that is available as Bioglass. Other materials that may be
employed include porous materials, such as, for example, controlled
pore glass beads. Any other materials known in the art that are
capable of having one or more functional groups, such as any of an
amino, carboxyl, thiol, or hydroxyl functional group, for example,
incorporated on its surface, are also contemplated.
[0068] Preferred solid supports include columns having a
polypeptide immobilized on a surface of the column. The surface may
be a wall of the column, and/or may be provided by beads packed
into the central space of the column.
[0069] The polypeptide may be immobilised on the solid support.
Examples of methods of immobilisation encompassed within the scope
of the present invention include: adsorption, covalent binding,
entrapment and membrane confinement. In a preferred embodiment of
the present invention the interaction between the polypeptide and
the matrix is substantially permanent. In a further preferred
embodiment of the present invention, the interaction between the
peptide and the matrix is suitably inert to ion-exchange
chromatography. In a preferred embodiment, the polypeptide is
attached to the surface of the solid support. It is understood that
a person skilled in the art would have a large array of options to
choose from to chemically and/or physically attach two entities to
each other. These options are all encompassed within the scope of
the present invention. In a preferred embodiment of the present
invention, the polypeptide is adsorbed to a solid support through
the interaction of biotin with streptavidin. In a representative
example of this particular embodiment, a molecule of biotin is
bonded covalently to the polypeptide, whereupon the
biotin-polypeptide conjugate binds to streptavidin, which in turn
has been covalently bonded to a solid support. In another
embodiment of the present invention, a spacer or linker moiety may
be used to connect the molecule of biotin with the polypeptide,
and/or the streptavidin with the matrix.
[0070] By contacting the GAG mixture with the solid support
GAG-polypeptide complexes are allowed to form. These are
partitioned from the remainder of the mixture by removing the
remainder of the mixture from the solid support, e.g. by washing
the solid support to elute non-bound materials. Where a column is
used as the solid support non-binding components of the GAG mixture
can be eluted from the column leaving the GAG-polypeptide complexes
bound to the column.
[0071] In the present invention, it is understood that certain
oligosaccharides may interact in a non-specific manner with the
polypeptide. In certain embodiments, oligosaccharide which
interacts with the polypeptide in a non-specific manner may be
included in, or excluded from the mixture of compounds enriched
with one or more GAGs that modulate the effect of a heparin-binding
factor. An example of a non-specific interaction is the temporary
confinement within a pocket of a suitably sized and/or shaped
molecule. Further it is understood that these oligosaccharides may
elute more slowly than those oligosaccharides that display no
interaction with the peptide at all. Furthermore it is understood
that the compounds that bind non-specifically may not require the
input of the same external stimulus to make them elute as for those
compounds that bind in a specific manner (for example through an
ionic interaction). The present invention is capable of separating
a mixture of oligosaccharides into those components of that mixture
that: bind in a specific manner to the polypeptide; those that bind
in a non-specific manner to the polypeptide; and those that do not
bind to the polypeptide. These designations are defined
operationally for each GAG-peptide pair.
[0072] By varying the conditions (e.g. salt concentration) present
at the surface of the solid support where binding of the GAG and
polypeptide occurs those GAGs having the highest affinity and/or
specificity for the heparin-binding domain can be selected.
[0073] GAGs may accordingly be obtained that have a high binding
affinity for a protein of interest and/or the heparin-binding
domain of the protein of interest. The binding affinity (K.sub.d)
may be chosen from one of: less than 10 .mu.M, less than 1 .mu.M,
less than 100 nM, less than 10 nM, less than 1 nM, less than 100
pM.
[0074] GAGs obtained by the methods of the invention may be useful
in a range of applications, in vitro and/or in vivo. The GAGs may
be provided for use in stimulation or inhibition of cell or tissue
growth and/or proliferation and/or differentiation either in cell
or tissue culture in vitro, or in cells or tissue in vivo.
[0075] The GAGs may be provided as a formulation for such purposes.
For example, culture media may be provided comprising a GAG
obtained by the method of the present invention.
[0076] Cells or tissues obtained from in vitro cell or tissue
culture in the presence of GAGs obtained by the method of the
present invention may be collected and implanted into a human or
animal patient in need of treatment. A method of implantation of
cells and/or tissues may therefore be provided, the method
comprising the steps of: [0077] (a) culturing cells and/or tissues
in vitro in contact with GAGs obtained by the method of the present
invention; [0078] (b) collecting the cells and/or tissues; [0079]
(c) implanting the cells and/or tissues into a human or animal
subject in need of treatment.
[0080] The cells may be cultured in part (a) in contact with the
GAGs for a period of time sufficient to allow growth, proliferation
or differentiation of the cells or tissues. For example, the period
of time may be chosen from: at least 5 days, at least 10 days, at
least 20 days, at least 30 days or at least 40 days.
[0081] In another embodiment the GAGs may be formulated for use in
a method of medical treatment, including the prevention or
treatment of injury or disease. A pharmaceutical composition or
medicament may be provided comprising the GAGs and a
pharmaceutically acceptable diluent, carrier or adjuvant. Such
pharmaceutical compositions or medicaments may be provided for the
prevention or treatment of injury or disease. The use of a GAG
obtained by the method of the present invention in the manufacture
of a medicament for the prevention or treatment of injury or
disease is also provided. Optionally, pharmaceutical compositions
and medicaments according to the present invention may also contain
the protein of interest having the heparin-binding domain to which
the GAG binds. In further embodiments the pharmaceutical
compositions and medicaments may further comprise stem cells, e.g.
mesenchymal stem cells.
[0082] Treatment of injury or disease may comprise the repair,
regeneration or replacement of cells or tissue, such as connective
tissue (e.g. bone, cartilage, muscle, fat, tendon or ligament). For
the repair of tissue, the pharmaceutical composition or medicament
comprising the GAG may be administered directly to the site of
injury or disease in order to stimulate the growth, proliferation
and/or differentiation of new tissue to effect a repair of the
injury or to cure or alleviate (e.g. provide relief to the symptoms
of) the disease condition. The repair or regeneration of the tissue
may be improved by combining stem cells in the pharmaceutical
composition or medicament.
[0083] For the replacement of tissue, GAGs may be contacted with
cells and/or tissue during in vitro culture of the cells and/or
tissue in order to generate cells and/or tissue for implantation at
the site of injury or disease in the patient. Implantation of cells
or tissue can be used to effect a repair of the injured or diseased
tissue in the patient by replacement of the injured or diseased
tissue. This may involve excision of injured/diseased tissue and
implantation of new tissue prepared by culture of cells and/or
tissue in contact with a GAG obtained by the method of the present
invention.
[0084] Pharmaceutical compositions and medicaments according to the
present invention may therefore comprise one of: [0085] (a) GAGs
obtained by the method of the invention; [0086] (b) GAGs obtained
by the method of the invention in combination with stem cells;
[0087] (c) GAGs obtained by the method of the invention in
combination with a protein containing the heparin-binding domain
bound by the GAG; [0088] (d) GAGs obtained by the method of the
invention in combination with stem cells and a protein containing
the heparin-binding domain bound by the GAG; [0089] (e) Tissues or
cells obtained from culture of cells or tissues in contact with
GAGs obtained by the method of the invention.
[0090] GAGs isolated according to the method of the present
invention may be used in the repair or regeneration of bodily
tissue, especially bone regeneration, neural regeneration, skeletal
tissue construction, the repair of cardio-vascular injuries and the
expansion and self-renewal of embryonic and adult stem cells.
Accordingly, the GAGs may be used to prevent or treat a wide range
of diseases and injuries, including osteoarthritis, cartilage
replacement, broken bones of any kind (e.g. spinal disc fusion
treatments, long bone breaks, cranial defects), critical or
non-union bone defect regeneration.
[0091] The use of GAGs according to the present invention in the
repair, regeneration or replacement of tissue may involve use in
wound healing, e.g. acceleration of wound healing, healing of scar
or bone tissue and tissue grafting.
[0092] In another aspect, the present invention provides a
biological scaffold comprising GAGs isolated by the method of the
present invention. In some embodiments, the biological scaffolds of
the present invention may be used in orthopaedic, vascular,
prosthetic, skin and corneal applications. The biological scaffolds
provided by the present invention include extended-release drug
delivery devices, tissue valves, tissue valve leaflets,
drug-eluting stents, vascular grafts, wound healing or skin grafts
and orthopaedic prostheses such as bone, ligament, tendon,
cartilage and muscle. In a preferred embodiment of the present
invention, the biological scaffold is a catheter wherein the inner
(and/or outer) surface comprises one or more GAG compounds attached
to the catheter.
[0093] In another aspect, the present invention provides one or
more GAGs isolated by the method of the present invention for use
as an adjuvant. In an especially preferred aspect of the present
invention, the adjuvant is an immune adjuvant.
[0094] In another aspect, the present invention provides
pharmaceutically acceptable formulations comprising a mixture of
compounds comprising one or more GAGs, said mixture being enriched
with respect to GAGs that modulate a heparin-binding factor.
[0095] In another aspect, the invention provides pharmaceutically
acceptable formulations comprising: [0096] (i) a mixture of
compounds comprising one or more GAGs, said mixture being enriched
with respect to GAGs that modulate a heparin-binding factor; and
[0097] (ii) the heparin-binding factor, for separate, simultaneous
or sequential administration. In a preferred embodiment the
formulation comprises the mixture of compounds comprising one or
more GAGs, said mixture being enriched with respect to GAGs that
modulate a heparin-binding factor and the heparin-binding factor in
intimate admixture, and is administered simultaneously to a patient
in need of treatment. In a further embodiment of the invention, the
formulation comprises the mixture of compounds comprising one or
more GAGs, said mixture being enriched with respect to GAGs that
modulate BMP-2 and BMP-2 in intimate admixture, and is administered
simultaneously to a patient in need of treatment.
[0098] In another aspect of the present invention a kit is provided
for use in the repair, or regeneration of tissue, said kit
comprising (i) a predetermined amount of a GAG having high affinity
for a protein having a heparin-binding domain, and (ii) a
predetermined amount of the protein having said heparin-binding
domain.
[0099] In preferred embodiments the GAG is HS/BMP2 and the protein
having the heparin-binding domain is BMP2.
[0100] The compounds of the enriched mixtures of the present
invention can be administered to a subject as a pharmaceutically
acceptable salt thereof. For example, base salts of the compounds
of the enriched mixtures of the present invention include, but are
not limited to, those formed with pharmaceutically acceptable
cations, such as sodium, potassium, lithium, calcium, magnesium,
ammonium and alkylammonium. The present invention includes within
its scope cationic salts, for example the sodium or potassium
salts.
[0101] It will be appreciated that the compounds of the enriched
mixtures of the present invention which bear a carboxylic acid
group may be delivered in the form of an administrable prodrug,
wherein the acid moiety is esterified (to have the form --CO2R').
The term "pro-drug" specifically relates to the conversion of the
--OR' group to a --OH group, or carboxylate anion therefrom, in
vivo. Accordingly, the prodrugs of the present invention may act to
enhance drug adsorption and/or drug delivery into cells. The in
vivo conversion of the prodrug may be facilitated either by
cellular enzymes such as lipases and esterases or by chemical
cleavage such as in vivo ester hydrolysis.
[0102] Medicaments and pharmaceutical compositions according to
aspects of the present invention may be formulated for
administration by a number of routes, including but not limited to,
injection at the site of disease or injury. The medicaments and
compositions may be formulated in fluid or solid form. Fluid
formulations may be formulated for administration by injection to a
selected region of the human or animal body.
[0103] Administration is preferably in a "therapeutically effective
amount", this being sufficient to show benefit to the individual.
The actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of the
injury or disease being treated. Prescription of treatment, e.g.
decisions on dosage etc, is within the responsibility of general
practitioners and other medical doctors, and typically takes
account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners. Examples
of the techniques and protocols mentioned above can be found in
Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub.
Lippincott, Williams & Wilkins.
[0104] In this specification a patient to be treated may be any
animal or human. The patient may be a non-human mammal, but is more
preferably a human patient. The patient may be male or female.
[0105] Methods according to the present invention may be performed
in vitro or in vivo, as indicated. The term "in vitro" is intended
to encompass procedures with cells in culture whereas the term "in
vivo" is intended to encompass procedures with intact
multi-cellular organisms.
Stem Cells
[0106] Cells contacted with GAGs obtained by the method of the
present invention include stem cells.
[0107] The stem cells cultured and described herein may be stem
cells of any kind. They may be totipotent or multipotent
(pluripotent). They may be embryonic or adult stem cells from any
tissue and may be hematopoietic stem cells, neural stem cells or
mesenchymal stem cells. Preferably they are adult stem cells. More
preferably they are adult mesenchymal stem cells, e.g. capable of
differentiation into connective tissue and/or bone cells such as
chondrocytes, osteoblasts, myocytes and adipocytes. The stem cells
may be obtained from any animal or human, e.g. non-human animals,
e.g. rabbit, guinea pig, rat, mouse or other rodent (including
cells from any animal in the order Rodentia), cat, dog, pig, sheep,
goat, cattle, horse, non-human primate or other non-human
vertebrate organism; and/or non-human mammalian animals; and/or
human. Optionally they are non-human.
[0108] In this specification, by stem cell is meant any cell type
that has the ability to divide (i.e. self-renew) and remain
totipotent or multipotent (pluripotent) and give rise to
specialized cells if so desired.
[0109] Stem cells cultured in the present invention may be obtained
or derived from existing cultures or directly from any adult,
embryonic or fetal tissue, including blood, bone marrow, skin,
epithelia or umbilical cord (a tissue that is normally
discarded).
[0110] The multipotency of stem cells may be determined by use of
suitable assays. Such assays may comprise detecting one or more
markers of pluripotency, e.g. alkaline phosphatase activity,
detection of RUNX2, osterix, collagen I, II, IV, VII, X,
osteopontin, Osteocalcin, BSPII, SOX9, Aggrecan, ALBP,
CCAAT/enhancer binding protein-.alpha. (C/EBP.alpha.), adipocyte
lipid binding protein (ALBP), alkaline phosphatise (ALP), bone
sialoprotein 2, (BSPII), Collagen2a1 (CoII2a) and SOX9.
[0111] Mesenchymal stem cells or human bone marrow stromal stem
cells are defined as pluripotent (multipotent) progenitor cells
with the ability to generate cartilage, bone, muscle, tendon,
ligament and fat. These primitive progenitors exist postnatally and
exhibit stem cell characteristics, namely low incidence and
extensive renewal potential. These properties in combination with
their developmental plasticity have generated tremendous interest
in the potential use of mesenchymal stem cells to replace damaged
tissues. In essence mesenchymal stem cells could be cultured to
expand their numbers then transplanted to the injured site or after
seeding in/on scaffolds to generate appropriate tissue
constructs.
[0112] Thus, an alternative approach for skeletal, muscular, tendon
and ligament repair is the selection, expansion and modulation of
the appropriate progenitor cells such as osteoprogenitor cells in
the case of bone in combination with a conductive or inductive
scaffolds to support and guide regeneration together with judicious
selection of specific tissue growth factors.
[0113] Human bone marrow mesenchymal stem cells can be isolated and
detected using selective markers, such as STRO-I, from a CD34+
fraction indicating their potential for marrow repopulation. These
cell surface markers are only found on the cell surface of
mesenchymal stem cells and are an indication of the cells
pluripotency.
[0114] In yet a further aspect of the present invention, a
pharmaceutical composition comprising stem cells generated by any
of the methods of the present invention, or fragments or products
thereof, is provided. The pharmaceutical composition useful in a
method of medical treatment. Suitable pharmaceutical compositions
may further comprise a pharmaceutically acceptable carrier,
adjuvant or diluent.
[0115] In another aspect of the present invention, stem cells
generated by any of the methods of the present invention may be
used in a method of medical treatment, preferably, a method of
medical treatment is provided comprising administering to an
individual in need of treatment a therapeutically effective amount
of said medicament or pharmaceutical composition.
[0116] Stem cells obtained through culture methods and techniques
according to this invention may be used to differentiate into
another cell type for use in a method of medical treatment. Thus,
the differentiated cell type may be derived from, and may be
considered as a product of, a stem cell obtained by the culture
methods and techniques described which has subsequently been
permitted to differentiate. Pharmaceutical compositions may be
provided comprising such differentiated cells, optionally together
with a pharmaceutically acceptable carrier, adjuvant or diluent.
Such pharmaceutical composition may be useful in a method of
medical treatment.
Glycosaminglycans
[0117] As used herein, the terms `glycosaminoglycan` and `GAG` are
used interchangeably and are understood to refer to the large
collection of molecules comprising an oligosaccharide, wherein one
or more of those conjoined saccharides possess an amino
substituent, or a derivative thereof. Examples of GAGs are
chondroitin sulfate, keratin sulfate, heparin, dermatan sulfate,
hyaluronate and heparan sulfate. Heparan sulfates are preferred
embodiments of the present invention.
[0118] As used herein, the term `GAG` also extends to encompass
those molecules that are GAG conjugates. An example of a GAG
conjugate is a proteoglycosaminoglycan (PGAG, proteoglycan) wherein
a peptidic component is covalently bound to an oligosaccharide
component.
[0119] In the present invention, it is understood that there are a
large number of sources of GAG compounds including natural,
synthetic or semi-synthetic. A preferred source of GAGs is
biological tissue. A preferred source of GAGs is a stem cell. An
especially preferred source of GAGs is a stem cell capable of
differentiating into a cell that corresponds to a tissue that will
be the subject of treatment. For example, GAGs can be sourced from
preosteoblasts for use in bone regeneration or skeletal tissue
construction. In an especially preferred embodiment of the present
invention, GAGs may be sourced from an immortalised cell line. In a
further preferred embodiment of the present invention, GAGs may be
sourced from an immortalised cell line which is grown in a
bioreactor. Another preferred source of GAGs is a synthetic source.
In this respect, GAGs may be obtained from the synthetic
elaboration of commercially available starting materials into more
complicated chemical form through techniques known, or conceivable,
to one skilled in the art. An example of such a commercially
available starting material is glucosamine. Another preferred
source of GAGs is a semi-synthetic source. In this respect,
synthetic elaboration of a natural starting material, which
possesses much of the complexity of the desired material, is
elaborated synthetically using techniques known, or conceivable, to
one skilled in the art. Examples of such a natural starting
material are chitin and dextran, and examples of the types of
synthetic steps that may elaborate that starting material, into a
GAG mixture suitable for use in the present invention, are amide
bond hydrolysis, oxidation and sulfation. Another example of a
semi-synthetic route to GAGs of the desired structure comprises the
synthetic interconversion of related GAGs to obtain GAGs suitable
for use in the present invention.
Heparan Sulphate (HS)
[0120] In preferred aspects of the invention the glycosaminoglycan
or proteoglycan is preferably a heparan sulfate.
[0121] Heparan sulfate proteoglycans (HSPGs) represent a highly
diverse subgroup of proteoglycans and are composed of heparan
sulfate glycosaminoglycan side chains covalently attached to a
protein backbone. The core protein exists in three major forms: a
secreted form known as perlecan, a form anchored in the plasma
membrane known as glypican, and a transmembrane form known as
syndecan. They are ubiquitous constituents of mammalian cell
surfaces and most extracellular matrices. There are other proteins
such as agrin, or the amyloid precursor protein, in which an HS
chain may be attached to less commonly found cores.
[0122] "Heparan Sulphate" ("Heparan sulfate" or "HS") is initially
synthesised in the Golgi apparatus as polysaccharides consisting of
tandem repeats of D-glucuronic acid (GlcA) and
N-acetyl-D-glucosamine (GlcNAc). The nascent polysaccharides may be
subsequently modified in a series of steps:
N-deacetylation/N-sulfation of GlcNAc, C5 epimerisation of GlcA to
iduronic acid (IdoA), O-sulphation at C2 of IdoA and GlcA,
O-sulphation at C6 of N-sulphoglucosamine (GlcNS) and occasional
O-sulphation at C3 of GlcNS. N-deacetylation/N-sulphation, 2-O-,
6-O- and 3-O-sulphation of HS are mediated by the specific action
of HS N-deacetylase/N-sulfotransferase (HSNDST), HS
2-O-sulfotransferase (HS2ST), HS 6-O-sulfotransferase (HS6ST) and
HS 3-O-sulfotransferase, respectively. At each of the modification
steps, only a fraction of the potential substrates are modified,
resulting in considerable sequence diversity. This structural
complexity of HS has made it difficult to determine its sequence
and to understand the relationship between HS structure and
function.
[0123] Heparan sulfate side chains consist of alternately arranged
D-glucuronic acid or L-iduronic acid and D-glucosamine, linked via
(1->4) glycosidic bonds. The glucosamine is often N-acetylated
or N-sulfated and both the uronic acid and the glucosamine may be
additionally O-sulfated. The specificity of a particular HSPG for a
particular binding partner is created by the specific pattern of
carboxyl, acetyl and sulfate groups attached to the glucosamine and
the uronic acid. In contrast to heparin, heparan sulfate contains
less N- and O-sulfate groups and more N-acetyl groups. The heparan
sulfate side chains are linked to a serine residue of the core
protein through a tetrasaccharide linkage
eglucuronosyl-.beta.-(1.fwdarw.3)-galactosyl-.beta.-(1.fwdarw.3)-galactos-
yl-.beta.-(1.fwdarw.4)-xylosyl-.beta.-1-O-(Serine)) region.
[0124] Both heparan sulfate chains and core protein may undergo a
series of modifications that may ultimately influence their
biological activity. Complexity of HS has been considered to
surpass that of nucleic acids (Lindahl et al, 1998, J. Biol. Chem.
273, 24979; Sugahara and Kitagawa, 2000, Curr. Opin. Struct. Biol.
10, 518). Variation in HS species arises from the synthesis of
non-random, highly sulfated sequences of sugar residues which are
separated by unsulfated regions of disaccharides containing
N-acetylated glucosamine. The initial conversion of
N-acetylglucosamine to N-sulfoglucosamine creates a focus for other
modifications, including epimerization of glucuronic acid to
iduronic acid and a complex pattern of O-sulfations on glucosamine
or iduronic acids. In addition, within the non-modified, low
sulfated, N-acetylated sequences, the hexuronate residues remain as
glucuronate, whereas in the highly sulfated N-sulfated regions, the
C-5 epimer iduronate predominates. This limits the number of
potential disaccharide variants possible in any given chain but not
the abundance of each. Most modifications occur in the N-sulfated
domains, or directly adjacent to them, so that in the mature chain
there are regions of high sulfation separated by domains of low
sulfation (Brickman et al. (1998), J. Biol. Chem. 273(8),
4350-4359, which is herein incorporated by reference in its
entirety).
[0125] It is hypothesized that the highly variable heparan sulfate
chains play key roles in the modulation of the action of a large
number of extracellular ligands, including regulation and
presentation of growth and adhesion factors to the cell, via a
complicated combination of autocrine, juxtacrine and paracrine
feedback loops, so controlling intracellular signaling and thereby
the differentiation of stem cells. For example, even though heparan
sulfate glycosaminoglycans may be genetically described (Alberts et
al. (1989) Garland Publishing, Inc, New York & London, pp. 804
and 805), heparan sulfate glycosaminoglycan species isolated from a
single source may differ in biological activity. As shown in
Brickman et al, 1998, Glycobiology 8, 463, two separate pools of
heparan sulfate glycosaminoglycans obtained from neuroepithelial
cells could specifically activate either FGF-1 or FGF-2, depending
on mitogenic status. Similarly, the capability of a heparan sulfate
(HS) to interact with either FGF-1 or FGF-2 is described in WO
96/23003. According to this patent application, a respective HS
capable of interacting with FGF-1 is obtainable from murine cells
at embryonic day from about 11 to about 13, whereas a HS capable of
interacting with FGF-2 is obtainable at embryonic day from about 8
to about 10.
[0126] As stated above HS structure is highly complex and variable
between HS. Indeed, the variation in HS structure is considered to
play an important part in contributing toward the different
activity of each HS in promoting cell growth and directing cell
differentiation. The structural complexity is considered to surpass
that of nucleic acids and although HS structure may be
characterised as a sequence of repeating disaccharide units having
specific and unique sulfation patterns at the present time no
standard sequencing technique equivalent to those available for
nucleic acid sequencing is available for determining HS sequence
structure. In the absence of simple methods for determining a
definitive HS sequence structure HS molecules are positively
identified and structurally characterised by skilled workers in the
field by a number of analytical techniques. These include one or a
combination of disaccharide analysis, tetrasaccharide analysis,
HPLC and molecular weight determination. These analytical
techniques are well known to and used by those of skill in the
art.
[0127] Two techniques for production of di- and tetra-saccharides
from HS include nitrous acid digestion and lyase digestion. A
description of one way of performing these digestion techniques is
provided below, purely by way of example, such description not
limiting the scope of the present invention.
Nitrous Acid Digestion
[0128] Nitrous acid based depolymerisation of heparan sulphate
leads to the eventual degradation of the carbohydrate chain into
its individual disaccharide components when taken to
completion.
[0129] For example, nitrous acid may be prepared by chilling 250
.mu.l of 0.5 M H.sub.2SO.sub.4| and 0.5 M Ba(NO.sub.2).sub.2
separately on ice for 15 min. After cooling, the Ba(NO.sub.2).sub.2
is combined with the H.sub.2SO.sub.4 and vortexed before being
centrifuged to remove the barium sulphate precipitate. 125 .mu.l of
HNO.sub.2 was added to GAG samples resuspended in 20 .mu.l of
H.sub.2O, and vortexed before being incubated for 15 min at
25.degree. C. with occasional mixing. After incubation, 1 M
Na.sub.2CO.sub.3 was added to the sample to bring it to pH 6. Next,
100 .mu.l of 0.25 M NaBH.sub.4 in 0.1 M NaOH is added to the sample
and the mixture heated to 50.degree. C. for 20 min. The mixture is
then cooled to 25.degree. C. and acidified glacial acetic acid
added to bring the sample to pH 3. The mixture is then neutralised
with 10 M NaOH and the volume decreased by freeze drying. Final
samples are run on a Bio-Gel P-2 column to separate di- and
tetrasaccharides to verify the degree of degradation.
Lyase Digestion
[0130] Heparinise III cleaves sugar chains at glucuronidic
linkages. The series of Heparinase enzymes (I, II and III) each
display relatively specific activity by depolymerising certain
heparan sulphate sequences at particular sulfation recognition
sites. Heparinase I cleaves HS chains with NS regions along the HS
chain. This leads to disruption of the sulphated domains.
Heparinase III depolymerises HS with the NA domains, resulting in
the separation of the carbohydrate chain into individual sulphated
domains. Heparinase II primarily cleaves in the NA/NS "shoulder"
domains of HS chains, where varying sulfation patterns are found.
Note: The repeating disaccharide backbone of the heparan polymer is
a uronic acid connected to the amino sugar glucosamine. "NS" means
the amino sugar is carrying a sulfate on the amino group enabling
sulfation of other groups at C2, C6 and C3. "NA" indicates that the
amino group is not sulphated and remains acetylated.
[0131] For example, for depolymerisation in the NA regions using
Heparinase III both enzyme and lyophilised HS samples are prepared
in a buffer containing 20 mM Tris-HCL, 0.1 mg/ml BSA and 4 mM
CaCl.sub.2 at pH 7.5. Purely by way of example, Heparinase III may
be added at 5 mU per 1 .mu.g of HS and incubated at 37.degree. C.
for 16 h before stopping the reaction by heating to 70.degree. C.
for 5 min.
[0132] Di- and tetrasaccharides may be eluted by column
chromatography.
Heparin-Binding Domains
[0133] Cardin and Weintraub (Molecular Modeling of
Protein-Glycosaminoglycan Interactions, Arteriosclerosis Vol. 9 No.
1 January/February 1989 p. 21-32), incorporated herein in entirety
by reference, describes consensus sequences for polypeptide
heparin-binding domains. The consensus sequence has either a
stretch of di- or tri-basic residues separated by two or three
hydropathic residues terminated by one or more basic residues. Two
particular consensus sequences were identified: XBBXBX [SEQ ID NO:
15] and XBBBXXBX [SEQ ID NO: 16] in which B is a basic residue
(e.g. Lysine, Arginine, Histidine) and X is a hydropathic residue
(e.g. Alanine, Glycine, Tyrosine, Serine). Heparin-binding domains
are reported to be abundant in amino acids Asn, Ser, Ala, Gly, Ile,
Leu and Tyr and have a low occurrence of amino acids Cys, Glu, Asp,
Met, Phe and Trp.
[0134] These consensus sequences may be used to search protein or
polypeptide amino acid sequences in order to identify candidate
heparin-binding domain amino acid sequences which may be
synthesised and tested for GAG binding in accordance with the
present invention.
[0135] WO 2005/014619 A2 also discloses numerous heparin-binding
peptides. The contents of WO 2005/014619 A2 are incorporated herein
in entirety by reference.
Proteins Containing Heparin-Binding Domains
[0136] The following proteins are known to contain heparin-binding
domains, and polypeptides derived from the amino acid sequences of
these proteins may be used for the identification of GAGs according
to the present invention.
Mitogens/Morphogens/Chemokines
[0137] Fibroblast Growth Factors (FGF-1, FGF-2, FGF-3, FGF-4,
FGF-5, FGF-6, FGF-7, FGF-8, FGF-9) as well as the FGF receptors
FGFR1, FGFR2, FGFR3; HGF (Hepatocyte growth factor); VEGFs
(Vascular endothelial growth factor); Activins; BMPs (Bone
morphogenetic protein, e.g. BMP-2, BMP-4); TGF-.beta.s
(Transforming growth factor); PDGFs (Platelet-derived growth
factor); OPG (Osteoprotegerin); HB-GAM (Heparin-binding
growth-associated molecules); pleiotropins; GM-CSF
(Granulocyte-macrophage colony-stimulating factor);
Interferon-.chi.; NT4/5 (Neurotophin); GDNF (Glial cell-derived
neurotrophic factor); Wnts Hedgehogs.
Antagonists
[0138] Noggin, Chordin, Sclerostin, CTGF (Connective Tissue Growth
Factor), Follistatin, Gremlin.
Adhesive Glycoproteins
[0139] Fibronectin, Vitronectin, Laminin, Collagens,
Thrombospondin, Tenascin, vonWillebrand Factor, NCAM (Neural Cell
Adhesion Molecule), N-cadherin
Enzymes
[0140] Lipoprotein Lipase, Hepatic Lipase, Phospholipase,
Apolipoprotein B, Apolipoprotein E.
Swine Protease Inhibitors
[0141] Antithrombin III, Heparin Co-factor II, Protease Nexins
Other Factors
[0142] Superoxide Dimustase, Elastase, Platelet Factor 4, N-CAM,
Transcription Factors, DNA Topoisomerase, RNA Polymerase, Tumor
Necrosis Factor.
[0143] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0144] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0145] Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are
incorporated herein by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0146] Embodiments and experiments illustrating the principles of
the invention will now be discussed with reference to the
accompanying figures in which:
[0147] FIG. 1. Anion exchange chromatography of MX samples
disrupted using 8 M Urea/CHAPS buffer. A large GAG peak is observed
after 1M NaCl elution.
[0148] FIG. 2. Representative chromatogram of the desalting system
during MX-derived GAG purification. The initial peak (12-18 min)
represents full length GAG chains. The conductivity peak and debris
peak (19-30 min) represent salt and GAG debris elution.
[0149] FIG. 3. tGAGs (2.5 mg) loaded onto an underivatised Hi-Trap
streptavidin column. All GAGs elute from the column in the
flowthrough, indicating no "background" attachment of GAGs to the
column.
[0150] FIG. 4. BMP2-HBP (1 mg) pre-incubated with tGAGs (25 mg) for
30 min. Elution profile shows the peptide (280 nm) exiting the
column in the flowthrough together with the tGAG sample.
[0151] FIG. 5. BMP2-HBP (1 mg) loaded onto a Hi-Trap column. The
280 nm absorbance levels indicate that the peptide remains attached
to the column even under high salt conditions; thus there was
successful coupling of the biotinylated peptide to the streptavidin
linker.
[0152] FIG. 6. BMP2-HBP (1 mg) coupled column loaded with of 25 mg
of tGAGs. The chromatogram (232 nm) clearly shows both an
overloading of the column, in the flow through as well as the
binding of some GAGs to the BMP2-HBP bed.
[0153] FIG. 7. Re-application of the GAG- (flowthrough) fractions
from the previous experiment (FIG. 6). The presence of a
significant GAG+ elution peak indicates that all available BMP2-HBP
binding sites had been saturated, resulting in a large proportion
of susceptible GAGs exiting the column in the flowthrough.
[0154] FIG. 8. BMP2-HBP (2 mg) coupled column loaded with tGAGs (6
mg). The chromatogram (232 nm) clearly shows no overloading of the
column, and the presence of a GAG subpopulation with a relative
affinity for the BMP2-HBP.
[0155] FIG. 9. Re-run of GAG- (flowthrough) from previous run (FIG.
8). The absence of a GAG+ elution peak indicates that the available
BMP2-HBP binding sites were not saturated in the previous run,
allowing the efficient extraction of GAG+ sugars in a single
run.
[0156] FIG. 10. Re-application of isolated full length GAG+
fractions (2 mg) shows no change in affinity for the BMP2-HBP (2
mg) column prior to heparinase III digestion. A reapplication of
GAG- fractions against the BMP2-HBP column also showed no change in
affinity, with all GAGs exiting the column in the flowthrough
essentially as in FIG. 9.
[0157] FIG. 11. GAG- fractions (1 mg) digested with heparinase III
before loading onto the BMP2-HBP (2 mg) column. The chromatogram
(232 nm) clearly shows that no GAG samples remain bound to the
column, but exit in the flowthrough. This indicates the absence of
any GAG+ domains in the full length GAG- chains.
[0158] FIG. 12. GAG+ fractions (2 mg) digested with heparinase 3
before loading onto the BMP2-HBP (2 mg) column. The chromatogram
(232 nm) demonstrates that the GAG+ samples are retained by the
column, suggesting that all domains on the full length GAG+ chain
have a relative affinity for the BMP2-HBP. The increase in the
absorbance peak, as compared to the same dry weight quantity of
GAG+ (FIG. 10), indicates the efficacy of the heparinase 3
treatment.
[0159] FIG. 13. Full length GAG+ chains separated using a Biogel
P10 column with an exclusion limit of between 1.5 kDa and 20 kDa.
The chromatogram shows that a large proportion of the sample chains
have an overall molecular weight of more than 20 kDa.
[0160] FIG. 14. Full length GAG+ sugar chains treated with nitrous
acid for 20 min to diagnostically degrade heparan sulfate species.
The chromatogram, generated from a Biogel P10 sizing column, shows
an almost complete degradation of all GAG+chains as compared to
FIG. 13, indicating that GAG+ isolated chains consist primarily of
heparan sulfate.
[0161] FIG. 15. Chondroitin-4-sulfate (6 mg) loaded onto BMP2-HBP
(2 mg) column. The chromatogram clearly illustrates a significant
proportion of the GAG chains having an affinity for the peptide, as
they eluted at a similar salt concentration as the GAG+samples.
[0162] FIG. 16. Chondroitin-6-sulfate (6 mg) loaded onto BMP2-HBP
(2 mg) column. The chromatogram indicates that few of the C6S GAG
chains have any affinity for the peptide column.
[0163] FIG. 17. Dermatan sulfate (6 mg) loaded onto the BMP2-HBP (2
mg) affinity column. The chromatogram indicates that few of the DS
GAG chains had any affinity for the peptide, with only a small
proportion of the GAGs being eluted at a similar salt concentration
to GAG+ samples.
[0164] FIG. 18. Bovine heparan sulfate (2.5 mg) loaded onto the
BMP2-HBP (2 mg) column. The chromatogram (232 nm) reveals only a
small fraction of the GAGs binding to the column.
[0165] FIG. 19. Heparin-LMW (50 mg) loaded onto the BMP2-HBP (2 mg)
column. The chromatogram (232 nm) reveals that almost no GAG bound
to the peptide.
[0166] FIG. 20. Heparin-HMW (28 mg) loaded onto the BMP2-HBP (2 mg)
column. The chromatogram (232 nm) reveals that almost no GAG bound
to the peptide.
[0167] FIG. 21. Heparin-HMW (25 mg) predigested with heparinase I
was loaded onto the BMP2-HBP (2 mg) column. The chromatogram (232
nm) reveals that very few GAG fragments bound to the peptide.
[0168] FIG. 22. Chromatogram showing steps in isolation of BMP-2
peptide specific HS by affinity chromatography.
[0169] FIG. 23. Chromatogram showing elution of BMP-2 peptide
specific HS (GAG+) by affinity chromatography.
[0170] FIG. 24. Chromatogram showing elution of BMP-2 peptide
non-specific HS (GAG-) by affinity chromatography.
[0171] FIG. 25. Chromatogram showing elution of Sigma HS(H9902)
standard under size exclusion chromatography on Superdex 75
column.
[0172] FIG. 26. Chromatogram showing elution of BMP-2 peptide
specific HS (GAG+) under size exclusion chromatography on Superdex
75 column.
[0173] FIG. 27. Graph showing Osterix expression in C2C12 cells in
response to control media, 100 ng/ml BMP2 and 300 ng/ml BMP2.
[0174] FIG. 28. Graph showing Osteocalcin expression in C2C12 cells
in response to control media, 100 ng/ml BMP2 and 300 ng/ml
BMP2.
[0175] FIG. 29. Graph showing Runx2 expression in C2C12 cells in
response to control media, 100 ng/ml BMP2 and 300 ng/ml BMP2.
[0176] FIG. 30. Graph showing expression of Alkaline Phosphatase as
measured by quantitative PCR in C2C12 cells in response to control
media, BMP-2, Negative GAG (GAG-), Positive GAGs (GAG+), Total HS
and Heparin (Hep).
[0177] FIG. 31. Graph showing expression of Osterix as measured by
quantitative PCR in C2C12 cells in response to control media,
BMP-2, Negative GAG (GAG-)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total
HS and Heparin (Hep).
[0178] FIG. 32. Graph showing expression of BspII as measured by
quantitative PCR in C2C12 cells in response to control media,
BMP-2, Negative GAG (GAG-)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total
HS and Heparin (Hep).
[0179] FIG. 33. Graph showing expression of Runx2 as measured by
quantitative PCR in C2C12 cells in response to control media,
BMP-2, Negative GAG (GAG-)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total
HS and Heparin (Hep).
[0180] FIG. 34. Graph showing expression of Osteocalcin in C2C12
cells in response to BMP and GAG+ (+BMP-2) isolated from MC3T3-E1
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0181] The details of one or more embodiments of the invention are
set forth in the accompanying description below including specific
details of the best mode contemplated by the inventors for carrying
out the invention, by way of example. It will be apparent to one
skilled in the art that the present invention may be practiced
without limitation to these specific details.
[0182] We investigated the potential of GAGs to augment the
activities of bone morphogenic protein 2 (BMP2). The highly
osteoinductive activity of BMP2 for the murine myogenic cell line
C2C12 have been well characterised. Studies both in this cell line,
and in vivo, have implicated a role for glycosaminoglycans in
modulating this activity.
[0183] BMP2's affinity for heparin has similarly been well
characterised. Numerous studies have been conducted that have
sought to examine the dynamic interaction between BMP2 and GAGs.
Some have proposed that the interaction is inhibitory, and so
responsible for either sequestering the cytokine away from the
receptor or inducing its association with its numerous inhibitors,
such as noggin, that have been shown, similarly, to have an
affinity for heparin. Alternative findings implicate the
interaction between BMP2 and GAGs is one of maintaining a local
concentration of the cytokine around cells that require its
signalling in order to differentiate into the osteoblast
lineage.
[0184] These findings also suggest that the association serves to
significantly lengthen the half-life of the homodimer, so allowing
it to remain active in the ECM for longer periods. As is the case
with most systems, the actual role of this interaction is likely to
be blend of some, or all of the above.
[0185] Although many studies have provided evidence for the
interaction that BMP2 has with model sugars, the specific
interaction between the BMP2 heparin-binding peptide (BMP2-HBP), a
string of amino acids (QAKHKQRKRLKSSCKRHP [SEQ ID NO: 1]) located
at the N-terminal end of each BMP2 monomer, and appropriate
glycosaminoglycans has received relatively little attention. A
major question that arises is whether there is a complementary
saccharide sequence embedded within an HS chain that controls the
association with an absolute, or at least relative,
specificity.
[0186] We sought to isolate a sequence-specific glycosaminoglycan
that could modulate BMP2 activity via a direct interaction with the
cytokine.
Example 1
Materials and Methods
Buffer Preparation
[0187] Preparation of all buffers for GAG extraction and analysis
is conducted with strict attention paid to quality. It is vital
that the pH of buffers is maintained at the correct level and that
all buffers be filtered and degassed in order to prevent the
clogging of columns with precipitates or bubbles. The formation of
bubbles, in particular, can cause serious damage to columns, and in
the case of sealed, pre-fabricated columns, leads to them becoming
unusable.
[0188] All buffers used were filtered with 1.times.PBS without
Ca.sup.2+ or Mg.sup.2+ (150 mM NaCl), or double distilled
(ddH.sub.2O) to make the final solutions.
Disruption Buffer
[0189] The 8M Urea/CHAPS disruption buffer consisted of PBS (150 mM
NaCl) with 1% CHAPS, 8M Urea and 0.02% NaN.sub.3 to prevent
contamination by microbial growth during storage. This solution was
used to disrupt matrix (MX) samples, so was not degassed or
filtered.
PGAG Anion Exchange Low Salt (250 mM) Buffer
[0190] Low salt PGAG anion exchange buffer comprised PBS (150 mM
NaCl) with an additional 100 mM NaCl. The buffer was equilibrated
to pH 7.3 with NaOH and 0.02% NaN.sub.3. The solution was then
degassed under negative pressure and constant stirring until no
further bubbles were released before being filtered through a 0.4
.mu.m filter.
PGAG Anion Exchange High Salt (1M) Buffer
[0191] High salt PGAG anion exchange buffer comprised PBS (150 mM
NaCl) with an additional 850 mM NaCl. The buffer was equilibrated
to pH 7.3 with NaOH and 0.02% NaN.sub.3 added. The solution was
then degassed under negative pressure and constant stirring before
being filtered through a 0.4 .mu.m filter.
Pronase/Neuraminidase PGAG Reconstitution Buffer
[0192] This buffer was used to reconstitute desalted PGAG samples
after anion exchange in order to prepare them for enzymatic
digestion of the associated core proteins. It consisted of 25 mM
sodium acetate (CH.sub.3COOHNa). The buffer was equilibrated to pH
5.0 with glacial acetic acid (CH.sub.3COOH). Both pronase and
neuraminidase enzymes were reconstituted according to the
manufacturer's instructions.
GAG Affinity Chromatography Low Salt (150 mM) Buffer
[0193] Low salt GAG anion exchange buffer was made using PBS (150
mM NaCl) without any additional salt. The buffer was equilibrated
to pH 7.3 with NaOH and 0.02% NaN.sub.3. The solution was degassed
under negative pressure and constant stirring until no further
bubbles were released before being filtered through a 0.4 .mu.m
filter.
GAG Affinity Chromatography High Salt (1M) Buffer
[0194] High salt GAG anion exchange buffer was made using PBS (150
mM NaCl) with an additional 850 mM NaCl. The buffer was
equilibrated to pH 7.3 with NaOH and 0.02% NaN.sub.3 was added, the
solution was then degassed and filtered through a 0.4 .mu.m
filter.
Desalting Solution
[0195] The desalting solution was made using ddH.sub.2O that was
equilibrated to pH 7.0 with 0.02% NaN.sub.3. The solution was then
degassed and filtered.
Sample Preparation
[0196] Matrix samples were disrupted using Disruption Buffer (8M
Urea/CHAPS), then scraped off the culture surface in this buffer
and stirred overnight at 37.degree. C. to ensure maximal lysis. The
samples were then centrifuged at 5000 g for 30 min and the
supernatant was clarified through a 0.4 .mu.m filter in preparation
for PGAG extraction via anion exchange chromatography.
Column Preparation & Usage
[0197] The choice and preparation of the types of columns to be
used for each sequential step in the isolation and characterisation
of GAGs is of major importance for the success of the protocol. It
was vital that at each step the columns were equilibrated and
cleaned with great care.
Anion Exchange Columns
[0198] Due to the relatively large quantities of MX substrate used
for GAG extraction, and the high load this places on the column
system, it was necessary to pack and prepare a large anion exchange
column manually, specifically for this study. Capto Q anion
exchange beads (Pharmacia) were packed into a Pharmacia XK 26
column (Pharmacia) to produce a column with a maximum loading
capacity of 500 ml of MX lysate per run.
[0199] Prior to use, both the column and all buffers were
equilibrated to room temperature for 30 min, before washing and
equilibrating the column in PGAG Anion Exchange Low Salt (250 mM)
Buffer for 30 min until all absorbance channels remained stable.
The clarified cell lysate was then passed through the column which
was again rinsed in 500 ml of low salt buffer to remove any
nonspecifically bound debris. PGAGs were then eluted using 250 ml
of PGAG Anion Exchange High Salt (1M) Buffer and lyophilised prior
to desalting. The column was then rinsed in low salt buffer and
returned to 4.degree. C. for storage.
Desalting Protocol
[0200] After PGAG/GAG isolation it was necessary to remove the high
amount of salt that accumulated in the sample during elution from
the column. For this step, all eluted samples of the same
experimental group were combined and loaded onto 4.times. Pharmacia
HiPrep.TM. 26/10 desalting columns. Prior to use, both the columns
and all solutions were equilibrated to room temperature for 30 min
before washing and equilibrating the column in Desalting Solution
for 30 min until all absorbance channels achieved stability.
Lyophilised samples were reconstituted in Desalting Solution in the
minimum possible volume that resulted in a clear solution. This
combination of columns permitted the loading of up to 60 ml of
sample. Those fractions eluting from the column first were
lyophilised and retained for further separation or cell culture
application. The columns were then rinsed in Desalting Solution and
returned to 4.degree. C. for storage.
BMP2-HBP Column Preparation
[0201] The isolation of GAGs carrying relative affinities for BMP2
was conducted using a BMP2-HBP column. Approximately 2 mg of
biotinylated BMP2-HBP was prepared in 1 ml of the GAG Affinity
Chromatography Low Salt (150 mM) Buffer. This amount was loaded
onto a HiTrap Streptavidin HP column (Pharmacia) and allowed to
attach to the column for 5 min. The column was then subjected to a
complete run cycle in the absence of GAGs. The column was washed in
13 ml of low salt buffer at a flow rate of 0.5 ml/min before being
subjected to 10 ml of GAG High salt buffer at 1 ml/min. Finally the
column was rinsed with 10 ml of low salt buffer. During this
process data was carefully monitored to ensure that no peptide
elution or column degradation was observed.
GAG+ Sample Isolation
[0202] Once the BMP2-HBP column had been prepared and tested for
stability under normal running conditions, it was ready to be used
for the separation of GAG+chains from tGAG (total GAG) samples.
tGAG samples (6 mg) were prepared in 3 ml of GAG affinity low salt
(150 mM) buffer and injected into a static loop for loading onto
the column. Prior to use both the BMP2-HBP column and all buffers
were equilibrated to room temperature for 30 min before washing and
equilibrating the column in low salt buffer for 30 min until all
absorbance channels were stable. The sample was then loaded onto
the column at 0.5 ml/min and the column and the sample rinsed in 10
ml of low salt buffer at 0.5 ml/min. Retained GAG+ samples were
subsequently recovered by elution with 10 ml of high salt (1 M)
buffer and lyophilised for desalting. The column was then rinsed in
10 ml of low salt buffer and stored at 4.degree. C.
Pronase/Neuraminidase Treatment
[0203] In order to isolate GAG chains from their core proteins,
they were digested using pronase and neuraminidase. Lyophilized
PGAG samples were resuspended in a minimum volume of 25 mM sodium
acetate (pH 5.0) and clarified by filtration through a 0.4 .mu.m
syringe filter. Total sample volume was dispensed into 10 ml glass
tubes in 500 .mu.l aliquots. 500 .mu.l of 1 mg/ml neuraminidase was
added and incubated for 4 h at 37.degree. C. After incubation 5 ml
of 100 mM Tris-acetate (pH 8.0) was added to each sample. An
additional 1.2 ml of 10 mg/ml pronase, reconstituted in 500 mM
Tris-acetate, 50 mM calcium acetate (pH 8.0), was added to each
sample and incubated for 24 h at 36.degree. C. After treatment all
volumes were combined and prepared for anion exchange processing by
centrifugation and filtration.
GAG Digestion Protocols
[0204] The analysis of GAGs, including their sulfated domain sizes
and relative sulfation levels, was carried out by using established
protocols including degradation by either nitrous acid or
lyases.
Nitrous Acid Digestion
[0205] Nitrous acid-based depolymerisation of heparan sulfate leads
to the eventual degradation of the carbohydrate chain into its
individual disaccharide components when taken to completion.
Nitrous acid was prepared by chilling 250 .mu.l of 0.5 M
H.sub.2SO.sub.4 and 0.5 M Ba(NO.sub.2).sub.2 separately on ice for
15 min. After cooling, the Ba(NO.sub.2).sub.2 was combined with the
H.sub.2SO.sub.4 and vortexed before being centrifuged to remove the
barium sulfate precipitate. 125 .mu.l of HNO.sub.2 was added to GAG
samples resuspended in 20 .mu.l of H.sub.2O, and vortexed before
being incubated for 15 min at 25.degree. C. with occasional mixing.
After incubation, 1 M Na.sub.2CO.sub.3 was added to the sample to
bring it to pH 6. Next, 100 .mu.l of 0.25 M NaBH.sub.4 in 0.1 M
NaOH was added to the sample and the mixture was heated to
50.degree. C. for 20 min. The mixture was then cooled to 25.degree.
C. and acidified with glacial acetic acid to pH 3 in the fume hood.
The mixture was then neutralised with 10 M NaOH and the volume was
then decreased by freeze drying. The final samples were run on a
Bio-Gel P-2 column to separate di- and tetrasaccharides to verify
degradation.
Heparinase III Digestion
[0206] Heparinase III is an enzyme that cleaves sugar chains at
glucuronidic linkages. The series of heparinase enzymes (I, II and
III) each display relatively specific activity by depolymerising
certain heparan sulfate sequences at particular sulfation
recognition sites. Heparinase I cleaves HS chains within NS regions
along the chain. This leads to the disruption of the sulfated
domains that are thought to carry most of the biological activity
of HS. Heparinase III depolymerises HS within the NA domains,
resulting in the separation of the carbohydrate chain into
individual sulfated domains. Lastly, Heparinase II primarily
cleaves in the NA/NS "shoulder" domains of HS chains, where varying
sulfation patterns are found.
[0207] In order to isolate potential active domains we focused on
the depolymerisation of GAG+ NA regions. Both the enzyme and
lyophilised HS samples were prepared in a buffer containing 20 mM
Tris-HCl, 0.1 mg/ml BSA and 4 mM CaCl.sub.2 at pH 7.5. The
concentration of heparinase III added to each sample is governed by
the relative quantity of HS components in the sample. Our analysis,
via nitrous acid depolymerisation, indicated that the GAG+ samples
consisted of predominantly HS; thus the enzyme was used at 5 mU per
1 .mu.g of HS. The sample was incubated at 37.degree. C. for 16 h
before the reaction was stopped by heating to 70.degree. C. for 5
min. The sample was then applied to the appropriate column system
for further analysis.
Cell Culture
GAG Production
[0208] In order to isolate GAG species representative of developing
osteoblasts, MC3T3 cells were grown in osteogenic conditions for 8
days. The cellular component was removed via incubation in a dilute
solution of 0.02 M ammonium hydroxide (NH.sub.4OH) at 25.degree. C.
for 5 min. After 5 min, NH.sub.4OH was removed by inversion of the
culture surfaces. Treated cultures were allowed to dry in a laminar
flow cabinet overnight. The following day the treated cultures were
washed three times with sterile PBS and allowed to dry in the
laminar flow cabinet. Prepared matrix cultures were then stored
under sterile conditions in 4.degree. C. until primary
proteoglycans were liberated via treatment with disruption buffer
and anion exchange chromatography.
BMP2-Specific GAG Bioactivity
[0209] C2C12 myoblasts were subcultured every 48 h, to a maximum of
15 passages, by plating at 1.3.times.10.sup.4 cells/cm.sup.2 in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
FCS. Osteogenic differentiation was induced at 2.times.10.sup.4
cells/cm.sup.2 in DMEM supplemented with 5% FCS, nominated
concentrations of recombinant human bone morphogenic protein-2
(rhBMP2) and glycosaminoglycan fractions with a positive or
negative affinity for rhBMP2 (GAG+ and GAG- respectively). rhBMP2
and GAG fractions were pre-incubated for 30 min at 25.degree. C.
prior to addition to their corresponding C2C12 cultures. The
cultures were permitted to grow under these conditions for 5 days,
with media for each condition being changed every 48 h, before mRNA
samples were extracted and prepared for RQ-PCR analysis. Real time
PCR for osteocalcin expression was conduced using the ABI Prism
7000.RTM. sequence detection system (PerkinElmer Life Sciences).
Primers and probes were designed using Primer Express software
(v2.1, PE Applied Biosystems). The target probe was redesigned to
incorporate LNA bases and labelled with BHQ-1 (Sigma-Proligo). The
ribosomal subunit gene 18S (VIC/TAMRA) was used as an endogenous
control, with each condition consisting of three repeats, each
tested in triplicate. The raw PCR data was analysed using the ABI
Sequence Detector software. Target gene expression values were
normalised to 18S expression prior to the calculation of relative
expression units (REUs).
Results
Anion Exchange Chromatography
[0210] In order to successfully extract GAGs from MX samples, it is
necessary to remove other matrix proteins that may contaminate the
sample. As GAGs constitute the most negatively charged molecules in
the ECM, this is most effectively accomplished with anion exchange
chromatography.
[0211] Samples were disrupted using 8M Urea/CHAPS buffer and loaded
onto the anion exchange column. Unwanted protein and ECM debris
were washed from the column and the negatively charged GAGs eluted
with 1 M NaCl. A typical chromatogram (FIG. 1) clearly shows the
flowthrough of a large amount of nonadherent debris, as well as the
clean and tight elution of a large quantity of GAGs from the MX
preparation. Thus not only does this result demonstrate the
purification of GAGs by this method, it also confirms the retention
of a large number of GAGs in the ECM after treatment with
NH.sub.4OH.
Desalting
[0212] Virtually all chromatography methods employed to purify and
analyse GAGs at various stages of processing require elution with
high-salt buffers. As high salt conditions interfere with
affinity-based chromatography, it is necessary to desalt samples
after each stage of processing. This process is generally completed
with size exclusion chromatography. Under these conditions larger
molecules, such as GAGs, exit the column before small molecules,
including the salt and small GAG debris. The separation of GAGs
from the contaminating salt can be followed on the resulting
chromatogram (FIG. 2) which also serves to confirm that the GAG
chains remained intact during the treatment process.
BMP2-HBP Column System
Column Preparation
[0213] Due to the prohibitive costs involved in creating a BMP2
growth factor column with commercially available reagents, we
instead utilised a biotinylated preparation of the known
heparin-binding domain of BMP2 (BMP2-HBP). This peptide was
immobilised on a Hi-Trap Streptavidin HP column (1 ml) in order to
specifically retain GAG chains with an affinity for the specific
heparin-binding domain peptide.
[0214] First we examined any background affinity the GAGs may have
had for the naked streptavidin column by running the total GAG
(tGAG) fraction against a column bed devoid of BMP2-HBP (FIG. 3).
Our results confirmed that our MX derived tGAG samples carried no
inherent affinity for the streptavidin column. We further
investigated two separate methods of exposing tGAGs to the BMP2-HBP
for the purpose of separating chains with a specific affinity. The
peptide was either pre-incubated for 30 min with 25 mg of tGAGs
prior to loading onto the streptavidin column, or was loaded first,
with the tGAGs being run through the column bed thereafter.
[0215] Pre-incubation of tGAGs with the BMP2-HBP revealed the
complete inability of the peptide to associate with the column
(FIG. 4), let alone mediate any isolation of specific GAGs. When
the peptide was loaded onto the column alone, however, its
association with the column was absolute, with effectively no
elution of peptide, even under 1 M salt conditions (FIG. 5). This
high affinity association indicates that the biotin-streptavidin
association is functioning correctly, and suggests a possible
inhibition of binding to the column, when loaded together with
tGAGs, due to steric hindrance.
Column Loading Capacities
[0216] As the proportion of tGAGs that were likely to have a
relative affinity for the BMP2-HBP was unknown, we first sought to
standardise the quantities of tGAGs loaded onto the peptide column
at each run for separation. Hi-Trap columns were prepared by
immobilising 1 mg of the BMP2-HBP for the extraction of tGAGs with
a specific affinity for the BMP2 heparin-binding site. This amount
was selected so as to maximise the quantity of available peptide
for future experiments should column stability become compromised
over time. Instability is a significant problem with peptide
columns, with corresponding impacts on consistency. Initial
attempts at loading of 25 mg of tGAGs onto a 1 mg BMP2-HBP coupled
column resulted in a clear overloading, as observed via absorbance
at 232 nm in the flowthrough (FIG. 6). Although a significant
elution peak was observed, tGAGs with affinity for the HBP were
lost in the flowthrough due to overloading. This was examined by
re-running the flowthrough through the peptide column (FIG. 7).
This resulted in a significant GAG+ (elution) peak, indicating that
the previous run had saturated the column.
[0217] Further optimisation led us to routinely load no more than 6
mg of tGAGs onto a 2 mg BMP2-HBP column. This, as evidenced by the
flowthrough peak (FIG. 8) and the absence of a positive-binding
fraction (FIG. 9), forestalled column overloading. The extraction
of those tGAGs with an affinity for the BMP-HBP from each sample
set in a single pass allowed us, in turn, to separate GAG+ and GAG-
fractions more efficiently.
GAG Domain Analysis
GAG+Chain Specificity
[0218] With the establishment of a standardised protocol, we were
able to reproducibly isolate GAG+ fractions for further
analysis.
[0219] Given the domain structure of heparan sulfate that mediates
the binding specificity for proteins, it is likely that
multi-domain GAG chains that bind to the column are in fact
composed of a large proportion of chain with little or no specific
affinity for BMP2. Similarly, it is possible that chains that
appeared GAG- may in fact contain domains that carry some affinity
for the BMP2-HBP. In order to examine these possibilities, it was
necessary to break down the GAG chains into their component domains
for more extensive examination.
[0220] The enzyme heparinase III (heparitinase I) cleaves HS chains
primarily in those areas flanking highly sulfated regions, thereby
liberating the highly charged, protein-associating domains that
bind susceptible growth factors, in this case the BMP2-HBP. Both
GAG+ and GAG- fractions were exposed to heparinase digestion,
although neither fraction showed any change in their affinity for
the BMP2-HBP (FIG. 10).
[0221] Heparinase III digestion of both full length GAG+ and GAG-
fractions was subsequently conducted, and both digested sample sets
subsequently loaded onto the BMP2-HBP column to assess retention
affinity.
[0222] The efficacy of the heparinase digestion was validated by
the increase in relative absorbance of samples of equal dry weight
after enzymatic digestion, as shown in FIGS. 10 and 12. As the
monitoring of GAG chains at 232 nm is via the sugar chain itself
and, in particular, unsaturated bonds, any cleavage along the
chain's length by heparinase III, resulting in unsaturated bonds of
HS fragments, leads to an increase in absorbance.
[0223] Interestingly, heparinase digestion of full length GAG-
chains yielded no fractions carrying any notable affinity for the
BMP2-HBP (FIG. 11). However, the digestion of full-length GAG+
samples similarly resulted in no fractions that lacked affinity for
the BMP2-HBP (FIG. 12). This result suggests that entire chains of
BMP-binding GAG are produced containing domain repeats that have a
specific affinity for the HBP. Alternatively, the HBP may not be
able to yield sufficient discrimination between GAG+ domains with
varying affinity under these minimalist conditions.
GAG+ Composition
Full Length GAG+ Sizing
[0224] In order to examine the composition of GAG+ fractions from
the BMP2-HBP column, we first examined their average size. This was
to ensure that we were actually separating GAG chains of reasonable
length, rather than small fragments not carrying any specific
affinity. Although any sizing of GAG chains is problematical, owing
to their relatively rigid rod-like conformation, a set of
assumptions invoking Stoke's radius and apparent sphericity can be
made.
[0225] Full length GAG+ samples were loaded onto Biogel P10 gel
filtration columns (1 cm.times.120 cm) with an exclusion limit of
between 20 kDa to 1.5 kDa. Absorbance measured at 232 nm indicated
a large proportion of GAG+ molecules had an overall apparent size
greater than 20 kDa (FIG. 13).
[0226] It has been posited that sugar chains must be longer than
approximately 10-14 rings in order to potentiate significant
biological activity for the FGF family of mitogens. In terms of
apparent molecular weight, a chain of 14 fully sulfated
disaccharides corresponds to approximately 8.7 kDa. As the majority
of chains found in the GAG+ samples show an apparent molecular
weight >20 kDa, it is reasonable to assume that the interaction
that they carry for the BMP2-HBP has some specific affinity and is
not the result of a general non-specific interaction.
GAG+ Sugar Species
[0227] There are five major glycosaminoglycan sugar families:
hyaluronan, keratan sulfate, dermatan sulfate, chondroitin sulfate
and heparan sulfate. Of these five, only heparan sulfate,
chondroitin sulfate and dermatan sulfate have the capacity to
generate variably sulfated domains that may code for specific
interactions with particular cytokines such as BMP2. The
identification of the type of sugar species isolated using the
BMP2-HBP column was of crucial importance for this study, and was
determined using a combination of diagnostic chemical and enzymatic
degradations. In particular, heparan sulfate, one of the major GAG
candidates for the interaction with BMP2, can be completely
degraded into its disaccharide components in the presence of
nitrous acid.
[0228] Thus, our HBP-retained GAG samples were incubated with
nitrous acid for 20 min prior to separation on a Biogel P10 sizing
column. Examination of the resulting chromatogram revealed an
almost complete degradation of all GAG+ sugar samples, as measured
by absorbance at 232 nm and 226 nm (FIG. 14). This result strongly
suggests that the full length sugar chains isolated specifically
against the BMP2-HBP consist primarily of heparan sulfate, as other
sugar chains are not affected by nitrous acid depolymerisation.
[0229] Although almost all the GAG+ chains could be degraded in
such a manner, a small peak was nevertheless observed at higher
molecular weights (>20 kDa). It can be postulated to consist of
chondroitin sulfates, of which CS-B (dermatan sulfate) and CS-E
(chondroitin-4,6-sulfate) demonstrate sulfation complexity akin to
heparan sulfates.
GAG Species Analysis
BMP2-HBP Specific GAGs (Alternative Species)
[0230] The degradation of full length GAG+ chains by exposure to
nitrous acid clearly indicated that the majority of GAG+ sugar
chains consisted of the heparan sulfate sugar species (FIG. 14).
The degradation of the GAG+ sample was not, however, complete as
was observed by the remnant peak in the high molecular weight
region. The presence of this peak points strongly to the
possibility of other species of sugar chains, such as chrondroitin
or dermatan sulfate. We next sought to examine the possible
affinity the other two sugar types may have for this cytokine by
first examining a variety of commercially available chondroitin and
dermatan sugars for their affinity to the BMP2-HBP column.
[0231] We tested chondroitin-4-sulfate (C4S), chondroitin-6-sulfate
(C6S) and dermatan sulfate (DS) by, in each instance, loading 6 mg
of the sugar onto the BMP2-HBP column under the same conditions
used to isolate GAG+ chains from MC3T3 matrix samples.
[0232] The chromatograms illustrating the affinity of each of the 3
sugar chain types showed that only C4S (FIG. 15) had any
significant affinity for the peptide. This affinity taken together
with the lack of affinity for the BMP2-HBP column observed for both
C6S (FIG. 16) and DS (FIG. 17) samples, appears to indicate that
C4S has a particular, potentially significant, interaction with the
BMP2 heparin-binding site.
[0233] As any potential interaction between chondroitin sulfate and
BMP2 has not yet been well characterised, these results led us to
question the validity of column chromatography as an accurate
monitor of the BMP2/heparan interaction. In order to further
explore the specificity of the interaction dynamic, we tested
several commercially available sugar species for their affinity to
the column. These included heparan sulfate, low molecular weight
heparin (Heparin-LMW), high molecular weight heparin (Heparin-HMW)
and Heparin-HMW treated with heparinase I.
[0234] Interestingly, none of these commercially available GAG
species appeared to demonstrate any specific interaction with the
peptide column. Heparan sulfate from bovine kidney had very little
affinity (FIG. 18), a behaviour that was further confirmed by its
inability to positively augment FGF2-mediated cell proliferation
(data not shown), as is observed in the presence of HS2. This
reduced ability of this GAG sample to bind the column may be as a
result of it being sold in a relatively unsulfated form.
[0235] None of the tested heparin samples showed even a minor
affinity for the column. This is of particular interest as BMP2
itself was historically first isolated using heparin columns. In
order to confirm this result, both LMW (FIG. 19) and HMW (FIG. 20)
heparin were tested; neither showed any appreciable affinity for
the column.
[0236] As we surmised that the relatively small BMP2-HBP peptide
may have had difficulty maintaining its association with the much
larger heparin molecules, we next predigested the heparin-HMW
samples using heparinase I. These smaller heparin-HMW fragments
were then run over the BMP2-HBP column; this treatment did not,
however, appear to improve the ability of any of the heparin
samples to bind the peptide column (FIG. 21).
[0237] This inability of the peptide column to show any specific
interaction with any of the various preparations of heparin was
somewhat unexpected, due to BMP2 conventionally being isolated via
heparin affinity. It is possible, however, that this may be as a
result of the reversing of the "receptor-ligand" order of
interaction; in this case the BMP2-HBP represented the fixed
"receptor" as opposed to the heparin that represented the "ligand",
or that the concentrations of BMP2-HBP or soluble heparin favour a
dissociated state that rapidly negates any affinity under flow/salt
stress.
CONCLUSIONS
[0238] The use of a preosteoblast-derived ECM substrate provided us
with a useful model for simulating the activity of natively
secreted, ECM-associated GAGs in relation to such osteoinduction.
Though numerous previous studies have examined the role that this
native interaction has in modulating the activity of BMP2, this has
usually been conducted at the level of the cytokine, rather than
with a view to exploring the sequence specificity of the
biomodulating GAGs.
[0239] Hence here we sought to exploit the availability of natively
secreted GAGs in the MX substrate and their potential for direct,
sequence-specific interaction and modulation of BMP2-induced C2C12
myoblast commitment to the osteogenic lineage.
Anion Exchange
[0240] The use of this particular standard and well characterised
protocol provided us with conclusive evidence for GAG accessibility
from the NH.sub.4OH-treated MX substrate. Our initial concerns were
centred around the harsh chemical treatment used to lyse the
cellular components of the ECM, and that this may have also
resulted in the stripping of the majority of GAGs from the ECM.
However, the significant, high affinity peak observed in the anion
exchange chromatogram clearly illustrates the retention of a large
quantity of GAGs within the MX substrate. While this particular
methodology does not allow for the identification of individual GAG
species, it does offer conclusive evidence of their presence in the
sample due to their being amongst the most negatively-charged
molecules secreted by cells.
BMP2-HBP Column System
[0241] Previous research into the functional role of the BMP2
heparin-binding peptide provided us with a useful tool to
investigate the potentially specific interaction that BMP2 has with
GAGs. This single string of amino acids, located at the N-terminus
of each BMP2 monomer, appears to be solely responsible for
mediating BMP2's affinity for GAGs.
[0242] We thus investigated the use of this region of the BMP2
molecule as a ligand "bait" in attempts to retain those GAG chains
that carried relative affinity for the cytokine. The use of the
BMP2-HBP in this manner resulted in a significant retention of HS
to the peptide column (GAG+).
Column Preparation
[0243] Using an N-terminal biotinylated HBP we prepared a BMP2-HBP
affinity chromatography column, and were able to successfully
retain GAG samples that were candidates for controlling the native
BMP2 homodimer. Initial preparations of the column highlighted some
interesting problems. Preparations of biotinylated BMP2-HBP that
were premixed with tGAGs showed an inability to bind to the column.
As later tests showed that the BMP2-HBP easily attached to the
streptavidin column when loaded on its own this result indicated
that the GAGs interfered with the ability of the peptide's
biotinylation site to associate with the streptavidin column. The
tGAGs themselves carried no affinity for the streptavidin,
indicating that the direct interaction with the BMP2-HBP, possibly
via steric hindrance, was responsible for this.
Column Optimisation
[0244] Without any direct information that would allow us to
estimate the binding capacities of GAG+ sugars in our samples, our
peptide column needed to be optimised to ensure that excessive
sample loading would not lead to column saturation and consequent
sample loss. This initially involved intentionally saturating the
column in order to examine the binding capacity of a known quantity
of BMP2-HBP. Even with a large quantity of tGAGs the peptide was
capable of retaining the majority of GAG+sugar chains. Under these
conditions as little as 1 mg of BMP2-HBP was able to completely
retain all GAG+ chains within two cycles. The column thus appeared
to "simulate" a true BMP2 growth factor column and provide an
extremely efficient way of extracting GAG+ samples.
[0245] The optimisation of peptide-based columns for specific GAG
isolation is a complex procedure that varies greatly depending on
the size and individual chemical characteristics of the protein
used. Previous studies, utilising FGF-1 and 2 growth factor columns
(Turnbull and Nurcombe, personal communication), also showed a
significant need for continual column maintenance and short viable
column life-spans. These studies demonstrate the laborious nature
of working with peptide columns and the care that must be taken to
correctly optimise this manner of system. Unfortunately, while
other systems for the analysis of specific protein-GAG interactions
exist, these generally lack the capacity to isolate sufficient
quantities of GAGs for further analysis, making them inappropriate
for our intended course of study.
GAG Domain Analysis
[0246] GAG sulfation patterns are, particularly in the case of
heparan sulfate (HS), frequently concentrated into domains of high
sulfation that are interspaced with regions of little sulfation.
This grouping of sulfation sites into domains is what provides
region-specific binding of ligands to the GAG chain, allowing a
single sugar molecule to potentially bind a variety of different
targets, and to stabilise the interaction between these, as is seen
in the FGF system. Exceptions to this proposed model for HS-ligand
interactions include the interaction between interferon gamma
(IFN.gamma.) and heparan sulfate. In this instance the interaction
between the GAG and IFN.gamma. leads to an increased potency of the
cytokine. IFN.gamma. that remains dissociated from local GAGs is
rapidly processed into an inactive form, thereby preventing its
signalling in inappropriate areas after diffusion. IFN.gamma. also
displays four separate heparin-binding domains, each with a
different sequence, a finding not unusual for heparin-binding
proteins. However, only two domains found immediately at the
C-terminus of the protein have been shown to mediate INF.gamma.'s
heparin-binding characteristics. Importantly, sequence analysis of
the HS sequence with specific affinity for these two IFN.gamma.
heparin-binding sites revealed an interesting difference in
comparison to the commonly observed model of HS-ligand interaction.
In this case, the sequence of HS responsible for the binding of
IFN.gamma. was found to be composed of a predominantly N-acetylated
region, carrying little sulfation. This region was flanked by two
small N-sulfated regions. This differs significantly with the
system observed in FGF, where sulfation patterns in NS domains are
responsible for mediating the interaction between FGF and HS. In
recent years, this type of interaction has been observed in
numerous other systems, such as PDGF, IL-8 and endostatin. The
discovery of this kind of interaction with HS, as observed in these
cytokines, may be able to explain the bioactivity observed in
hyaluronan, which carries no sulfation patterns at any point along
its chain and yet has the ability to modulate the activity of such
factors as NF-.kappa.B.
[0247] These observed interactions between ligands and GAGs, in
particular that of IFN.gamma., differ significantly to the
proposed, and our observed, mode of interaction between HS and
BMP2. BMP2's single, N-terminal heparin-binding domain exhibits no
secondary structure and appears to interact with HS solely on the
basis of charge. While in-depth sequence analysis of HS that binds
this peptide sequence was not conducted, its requirement to be
eluted under approximately 300 mM NaCl conditions lead us to
suspect the presence of a moderate degree of sulfation, thereby
placing this interaction within the conventional model of sulfation
patterns mediating specific interactions.
GAG+ Chain Specificity
[0248] The allocation of sulfation patterns into domains that give
HS its ability to stabilise proteomic interactions also results in
the possibility that a GAG+ sugar chain of sufficient length and
complexity may carry several domains that have no direct affinity
for the BMP2-HBP on their own, due to their carrying a different
sulfation sequence. Conversely, it is also possible that some
full-length sugar chains that were identified as having little
affinity for the BMP2-HBP (GAG-) may contain some cryptic domains
that do carry such affinity.
[0249] In recent years, numerous reports have been published that
provide strong evidence for a "sulfation code" within these complex
carbohydrate chains. While the details of this "sulfation code"
remain difficult to elucidate, and the sequencing of long chains of
sulfated carbohydrates is a complex and time consuming process, a
number of possible modes of specific interaction between GAGs and
ligands have been proposed. One observation in particular has led
to the characterisation of numerous GAG-ligand models; the grouping
of sulfation into discrete regions, or "domains", along the length
of many types of GAGs, such as heparan sulfate. Interestingly no
template for this phenomenon has yet been observed, and it appears
to be primarily a result of the temporal activity of the
sulfotransferase enzymes responsible for this phase of GAG
synthesis.
[0250] Particularly useful tools in the study of specific GAG
sequences are a number of heparin lyases that can be used to
examine targeted depolymerisation of complex carbohydrate chains,
thereby providing insight into their structure. One particular
heparan lyase, heparinase III (heparitinase), cleaves heparin
sulfate chains at sites flanking the highly sulfated domains that
may occur in heparan sulfate chains. Thus, using this enzyme, it is
possible to liberate these potentially active regions from the full
length sugar chains and separate them, if they function as single
domains, via affinity chromatography, from regions with no specific
affinity for the BMP2-HBP.
[0251] It is important to note that, in the case of GAG-ligand
interactions, affinity by sequence does not necessarily guarantee
bioactivity. The mode of activity mediated by GAGs during their
association with their various ligands differs greatly depending on
the system. In some instances where the sugar chain is responsible
for prolonging protein-protein interaction via stabilisation of
tertiary protein structures, such as is found between FGF and its
receptor, and the interaction between HGF/SF and Met, multiple
discrete sulfation regions may be involved in mediating the
intended bioactivity of the sugar chain. In such instances the
isolation of individual sulfated domains from a full length
carbohydrate chain may, in fact, result in an inhibition of sugar
bioactivity since though each "domain-fragment" still binds its
intended target it is unable to mediate the intended biological
effect of a combined full length carbohydrate chain.
[0252] Interestingly, this particular characteristic of GAG-ligand
interactions is precisely what makes this manner of approach useful
for modulating BMP2 activity. The proposed model for GAG modulation
of BMP2 bioactivity involves immobilization of the cytokine to GAGs
in the ECM or on the cell surfaces. In this type of system the
application of exogenous GAGs specific to the heparin-binding
domain of BMP2 would prevent this interaction, increasing short
term BMP2 mediated signalling, similar to the effect observed
during the addition of soluble heparin. While there is some
indication that this manner of interaction would continue to
protect the cytokine from proteolytic degradation, delocalization
of BMP2 from its intended region of bioactivity has the potential
to negatively impact the cytokines effectiveness in the long
term.
[0253] Control testing of our full length GAG+ and GAG- chains
resulted in similar profiles to those observed during their primary
separation. Analysis of GAG+ and GAG- chains post treatment with
heparinase III, however, gave surprising results. The digestion of
GAG+ chains did not seem to generate separable fragments based on
simple affinity for the BMP2-HBP. Furthermore, the digestion of
full length GAG- chains yielded no liberation of positive domains
from the negative sugar chains. There is some possibility that the
enzymatic digestion did not go to completion. However, the
resulting chromatogram clearly showed a large increase in the
absorbance at 232 nm when compared to the full length GAG chains.
As a large proportion of the absorbance of glycosaminoglycans at
232 nm is mediated via absorbance of unsaturated bonds, such as
those formed during enzymatic depolymerisation, it strongly
indicates that the enzymatic digestion was, in fact,
successful.
[0254] The implications of this result are somewhat unusual. This
data suggests that GAG chains are not only synthesised by cells to
specifically interact with BMP2, but that, in the case of MC3T3
cells, these sugar chains carry a number of sequence repeats
specific for aspects of BMP2 metabolism. The fact that BMP2 is an
extremely potent factor may offer an explanation for this
observation. The effects of BMP2 on the osteoinduction of
mesenchymal progenitor cells is well documented, as is its ability
to induce ectopic bone formation in cells that are even more
removed from the osteogenic lineage. Given this potency, aberrant
signalling of BMP2 is known to have deleterious consequences both
for healing and in development. It is possible that numerous
repeats of the BMP2-HBP interaction sequence on preosteoblast GAGs
are designed to ensure a maximal binding, and thereby the
modulation, of this cytokine's ability to induce altered cell fate.
Conversely, the extremely low concentrations of BMP2 produced in
vivo may also require this type of sugar chain production in order
to ensure the retention of a sufficient local concentration, an
observation supported by the extremely high concentrations of BMP2
required in vitro to induce the osteogenic differentiation of C2C12
myoblast cells.
[0255] Of particular interest is the fact that this repetition of
BMP2-binding domains is produced via a synthesis pathway for which
no template or timing mechanism has yet been elucidated. The
accuracy and reproducibility of sequence specific domains within a
single sugar chain (as opposed to the random clustering of such
domains with those against other ligands) strongly suggests that
these cells do, in fact, have the ability to direct the generation
of specific sugar sequences. The current understanding of HS
structure implicates the progressive post-synthesis "editing" of
the carbohydrate chain in the generation of sequence-specific
regions, with observations pointing towards some manner of
enzymatic "template", whereby the local concentrations of
particular sulfotransferases as well as other interacting molecules
are used to directly control the generation of specific sugar
sequences. Our current understanding of this mode of specific
synthesis is largely formulated based on numerous studies including
those by Lindahl et al. that investigated the high affinity
interaction between antithrombin III and heparin, and those by Esko
et al. involving Chinese hamster ovary (CHO) cell mutants with
altered GAG synthesis pathways. These studies, while varying
significantly in their approaches to GAG analysis, all point
towards a highly conserved system of specific GAG synthesis, for
the directed modulation of cytokine and receptor activity.
Importantly, these studies also serve to explain the potential
generation of such BMP2 repeats as were observed in our study.
GAG+ Constitution
Full Length GAG+ Size
[0256] The bioactivity of individual GAGs chains for FGFs is
closely related to carbohydrate chain length. A common approach to
assessing GAG bioactivity is to assay ever shorter sulfated domain
fragments and so determine the shortest possible sequence required
to mediate the activity observed.
[0257] Using this approach we first examined full length GAG+ sugar
chains, and determined that they were >20 KDa in size, long
enough to carry multiple domains with affinity for BMP2.
Interestingly, this observation provided support for the earlier
observation that GAG+ samples treated with heparinase 3 showed
multiple repeats of carbohydrate chain segments with a specific
affinity for BMP2, since a variably sulfated sugar chain of this
size has the capacity to carry numerous sulfated domains.
GAG+ Sugar Species
[0258] With the majority of the five glycosaminoglycan types that
constitute the "glycome" able to encode the observed specific
interactions with BMP2, it was necessary to elucidate which of
these GAG types could be involved in this specific association.
Although the prime candidate for this interaction is a heparan
sulfate, analogous growth factor interactions have also been
identified for chondroitin and dermatan sulfates.
[0259] Heparan sulfate can be totally depolymerised into its
disaccharide components with nitrous acid. This particular
characteristic, shared with heparin and keratan sulfate, is
essential for the analysis of specific GAG populations. In the case
of our analysis of the carbohydrate constituents of our GAG+
samples, degradation due to nitrous acid was diagnostic of heparan
sulfate. This probability is primarily due to its heparan sulfate's
higher degree of charge patterning via sulfation in comparison to
either heparin or keratan sulfate. Ultimately, this charge
patterning is responsible for BMP2's specific interaction with
HS.
[0260] Our analysis utilising the nitrous acid protocol showed a
complete degradation of the GAG+ sample set indicating that the
majority of sugars in the GAG+ sample set were in fact 1,3-linked
and, thus, were heparan sulfate. This result supports the numerous
observations in regards to the specificity of heparan sulfate
cytokine interactions, particularly the interaction that BMP2
exhibits with heparin and HS.
GAG Species Analysis
BMP2-HBP Specific GAGs (Alternative Species)
[0261] The small remnant peak that was observed after the
degradation of GAG+ samples by nitrous acid supports the
possibility that other sulfated GAGs carrying some specific
affinity for BMP2 may be found in the GAG+ sample set. Given our
current understanding of the role of sulfation in mediating the
interaction between GAGs and BMP2, chondroitins and dermatans are
the most likely alternative sugars to show a specific interaction
with BMP2 as these show the highest potential diversity in
sulfation patterns.
[0262] A methodology frequently employed for GAG analysis includes
examining the role of individual sulfation positions on GAG-ligand
interactions. This method of analysis gives an indication of the
importance of individual sulfation positions in maintaining the
interaction between the GAG chain and its specific target.
Furthermore, since the different species of GAGs only have the
potential to carry sulfation patterns specific to their species,
this can aid in narrowing the possible glycosaminoglycan candidates
that may show an affinity for a specific ligand.
[0263] To this end we examined the affinity for the BMP2-HBP
carried by variably sulfated CS chains, C4S and C6S, and standard
DS. Interestingly, only C4S carried any significant affinity for
the BMP2-HBP. This data indicates that it is likely that the
4-O-sulfation is necessary for CS to interact with the BMP2-HBP.
Interestingly, dermatan sulfate showed no affinity for the
BMP2-HBP. This observation is of interest since DS is the only CS
species that demonstrates diversity in sulfation similar to that of
HS. Furthermore, our observations indicate a possibility that the
epimerisation of GlcA to IdoA in DS compromises the ability of this
sugar type to bind the BMP2-HBP. Both C4S and DS are able to carry
4-O-sulfation, yet only small quantities of DS were retained on the
column in comparison to C4S. Alternatively, this lack of affinity
may simply be due to this particular batch of DS not carrying
sufficient 4-O-sulfation to effectively mediate binding to the
BMP2-HBP. Interestingly, these particular observations appear to
demonstrate an interaction between BMP2 and CS carrying
4-O-sulfation. While previous studies have investigated the use of
CS-BMP2 interactions in drug delivery systems, not much is known
about any sequence specific interaction between individual CS
species and BMP2. However, since HS chains are composed of
1,4-linked disaccharide units, the observed 4-O-sulfation
responsible for CS-BMP2 interactions is not found in HS-BMP2
interactions, pointing to a sequence specific interaction not found
in CS. Thus it is likely that the remnant peak observed
post-nitrous acid treatment may contain small quantities of
4-O-sulfate carrying C4S or DS.
[0264] Further investigation revealed that neither commercial HS
nor heparin held any significant affinity for the peptide column.
The HS used for this assay was purchased commercially from
Sigma-Aldrich and was derived from bovine kidney. Given what is
known about the tissue specificity of HS it is possible that this
commercially available HS, isolated from bovine kidney sources,
carried negligible carbohydrate sequences required to specifically
mediate an interaction with BMP2. Similarly neither LMW nor HMW
heparin showed any affinity for the peptide column. The heparin
used for this analysis was also purchased from Sigma-Aldrich, and
was derived from porcine intestinal mucosa.
[0265] While heparin's interaction with antithrombin III has been
well characterised, and notwithstanding its versatile role in the
isolation of susceptible molecules, heparin's interaction with
growth factors is not, in general, regarded to be specific due to
its uniform sulfation. However, given that heparin is routinely
used to isolate BMP2, it is somewhat surprising that neither of the
heparin samples interacted with the peptide column to any
significant degree.
[0266] A further possibility for this lack of interaction between
the peptide column and heparin is due to the difference in
molecular weights between the two molecules. The small BMP2-HBP
attached to the column may have difficulty in maintaining its
association with the larger, heavily sulfated heparin chain. The
inability of heparinase-cleaved heparin to bind the column,
however, appeared to indicate that the steric effects of using full
length heparin on the column were not solely responsible for
disrupting the potential interaction between the sugars and the
BMP2-HBP. There is no immediately apparent reason for this
inability for commercial heparin to associate with the BMP2-HBP
column, though it may be postulated that further spatial separation
of the BMP2-HBP from its associated bead via spacer chains may help
to ameliorate this problem.
SUMMARY
[0267] In this study we have demonstrated the use of affinity
chromatography to isolate a subset of glycosaminoglycans that carry
a specific affinity for the BMP2-HBP, and have shown the potential
for this procedure to yield reproducible results. During this
portion of our investigation into the interaction between matrix
based GAGs and BMP2, we have made several observations with regards
to both the type of GAGs involved in mediating this association and
their structure.
[0268] Our results have implicated heparan sulfate for mediating
the majority of the affinity BMP2 has for the preosteoblast ECM, an
interaction which is increasingly recognised as being responsible
for the modulation of BMP2 activity. Furthermore, our investigation
into the likely structure of the ECM-resident GAGs isolated on the
basis of their affinity for the BMP2 heparin-binding site have
yielded a surprising result.
[0269] Our data indicates that full length BMP2 GAG+ chains do not
consist of individual domains with specific affinity for BMP2
interspersed with regions of little or no affinity for the factor.
Instead, our results imply that these GAG+ chains consist of
multiple BMP2-binding domain repeats. This result is surprising on
several levels. Firstly, the repetition required to fulfil this
observation over the full length of a >20 kDa carbohydrate chain
points to the presence of some manner of synthetic template.
Indeed, while previous studies have been unable to derive a
template for the assembly of tissue-specific GAG chains, the very
fact that such specificity exists supports the presence of a
template-based system. Although no genomic template has been
elucidated for this process there exists some possibility of a
proteomic, perhaps enzymatic, template.
[0270] Secondly, this observation provides some evidence as to the
importance of the interaction between BMP2 and GAGs. Multiple
repeats of the BMP2 affinity site along the length of the
carbohydrate chain may be required to ensure maximal binding of
BMP2 to the ECM. This particular association has been shown to
significantly lengthen the factor's half life, as well as probably
being responsible for maintaining a significant local concentration
in order to maintain signalling. Alternatively, some studies have
proposed a model whereby BMP2 is spatially inhibited from
interacting with its receptors due to the interactions with
ECM-based GAGs. In this particular scenario the repetition of BMP2
affinity sequences would ensure a maximal binding of the factor,
thus reducing the chance of it interacting with its receptors.
[0271] Our cumulative results indicated that this system for the
isolation of GAGs from the ECM is viable and likely to yield GAG
chains that have a specific affinity for BMP2.
[0272] This study supports previous findings in regards to the
interaction between GAGs and BMP2. Although the prevention of BMP2
associating with the ECM in vitro through the addition of exogenous
GAG+ appears to increase BMP2 signalling and upregulates osteogenic
gene expression, observations to the contrary have also reported.
In these studies, in vivo examination of BMP2's modulation via the
HBP showed a distinct improvement in long term osteogenesis when
the association with ECM GAGs was increased. It is possible that
this interaction plays a major role in maintaining local
concentrations by preventing the factor from diffusing away from
its sites of primary activity. In light of these studies and our
own observations, we propose that BMP2's activity is both
positively and negatively regulated by its association with GAGs.
Negative regulation may occur precisely via the model proposed by
Katagiri and colleagues, whereby the retention of BMP2 in the ECM,
away from its receptors, leads to a downregulation of BMP2
signalling. However, cells that require signalling by this factor
may potentially secrete various enzymes to remodel extracellular
sugar chains, such as sulfatases and heparinases, in order to "clip
away" GAGs retaining BMP2 in the ECM, thereby liberating the factor
and allowing it to signal, leading to the BMP2-ECM interaction
ultimately becoming one of positive maintenance of the cytokine's
activity. Alternatively, negative regulation of BMP2 by cell
surface GAGs, may be via the internalisation of GAG chains with
their associated BMP2 molecules, as has been observed by Jiao and
colleagues.
[0273] These previous studies, in conjunction with our own
observations, have lead us to conclude that the sequence-specific
interplay between BMP2 and heparin sulfate represents an intricate
control mechanism that has the capacity to both positively and
negatively regulate BMP2 signaling. Physiologically this
interaction is responsible for enforcing context dependent
responses to this potent cytokine in respect to many facets of
embryonic development, precursor commitment and wound healing.
Example 2
Purification of BMP2 Peptide Specific HS
[0274] We used a peptide having heparin-binding properties from the
mature BMP-2 sequence to identify novel HS that bind to the
peptide.
TABLE-US-00002 Mature BMP-2 amino acid sequence: [SEQ ID NO: 14]
QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFP
LADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVL KNYQDMVVEGCGCR
Heparin-binding peptide amino acid sequence: [SEQ ID NO: 1]
QAKHKQRKRLKSSCKRHP
[0275] To replicate the natural presentation of the heparin-binding
site we biotinylated the peptide on it's C-terminus and kept the
proline (P) to improve the flexibility/accessibility of the peptide
once bound to the streptavidin column.
Isolation of BMP2 Peptide Specific HS
[0276] Materials used included a BMP2--peptide coupled Streptavidin
column, HiPrep Desalting Column (GE Healthcare), 20 mM PBS+150 mM
NaCl (Low Salt Buffer), 20 mM PBS+ 1.5 M NaCl (High Salt Buffer),
HPLC grade Water (Sigma), Biologic-Duoflow Chromatography system
(Bio-Rad) and a Freeze Drier.
[0277] The column was equilibrated with Low Salt buffer and 1 mg
Sigma HS(H9902) was dissolved in low salt buffer and passed through
the BMP2-Streptavidin column. Unbound media components were removed
from the column by washing low salt buffer (20 mM PBS, pH 7.2, 150
mM NaCl) until the absorbance of the effluent at 232 nm almost
return to zero. HS bound to the matrix was eluted with high salt
buffer (20 mM PBS, pH 7.2, 1.5 M NaCl). Peak fractions were pooled
and freeze dried for 48 hrs.
[0278] HS 1 mg was applied to the column and washed with 20 mM PBS
buffer containing a low (150 mM) NaCl concentration. After washing
with low salt buffer, the bound HS were eluted with 20 mM PBS
buffer containing a high (1.5 M) NaCl concentration. Peaks
representing retained fractions (monitored at 232 nm) were
collected and subjected to further desalting.
[0279] After freeze drying 6 mg of positive HS (GAG+) and 1.8 mg of
negative HS (GAG-) were obtained.
Example 3
Evaluation of BMP-2 Specific Heparan Sulfates
[0280] C2C12 are mouse mesenchymal stem cells normally exhibiting
myogenic differentiation but capable of being directed in the
osteogenic lineage with supplementation of BMP-2 at passage 3.
C2C12 cells at passage 3 were maintained in DMEM with 1000 g/L
glucose (low glucose), 10% of FCS, 1% of P/S and without
L-glutamine (maintenance media).
[0281] DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of
P/S and without L-glutamine was used as differentiation media.
Effect of BMP-2 on Osteogenesis
[0282] We evaluated the effects of exogenous BMP-2 on osteogenesis
by measuring the levels of expression of osteogenic markers
(osteocalcin, osterix, Runx2).
[0283] Through assaying the effect of addition of different amounts
(100 ng/ml and 300 ng/ml) of BMP-2 to the cells we observed a
significant decrease at day 5 in the expression of osterix,
osteocalcin and Runx2 in cells having 100 ng/ml BMP-2 compared to
addition of 300 ng/ml BMP-2 (FIGS. 27-29). Thus we chose this time
point for future tests, as any changes should be readily
observable.
Materials and Methods
[0284] C2C12 cells at passage 3 were used. Cells were kept in
liquid Nitrogen at Passage 3 with 1.times.10.sup.6 cells/vial. Once
cells were taken from liquid Nitrogen, we added 500 .mu.l of
culture media, pipetted up and down to refreeze the cells and
immediately added 15 ml of culture media.
[0285] Culture media was DMEM with 1000 g/L glucose (low glucose),
10% of FCS, 1% of P/S and without L-glutamine. Treatment media was
DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of P/S and
without L-glutamine.
[0286] C2C12 cells were allowed to grow to 75% confluence before
harvesting (normally 2 to 3 days) in culture media.
[0287] Cells were counted as follows. Media was first
aspirated/discarded; 15 ml of PBS added, discard the PBS and add 3
ml of trypsin, incubate at 37.degree. C. for 5 min to lift the
cells from the flask. 9 ml of culture media added to neutralize the
trypsin. GUAVA used to determine the amount of cells for subsequent
cell seeding onto the experiment plates. For example, for 3 sets of
12-well plates 30,000 cells.times.36 wells.times.3.7
cm.sup.2=4,000,000 cells. Dilute the cells from the stock and add
the desired amount of culture media for cell seeding (each well
requiring 500 .mu.l of media with 30,000 cells).
[0288] To prepare BMP2 stock 10 .mu.g rhBMP2 (Bone Morphogenetic
Protein 2) was re-suspended in 100 .mu.l of 4 mM HCl/0.1% BSA.
[0289] The following RNA extraction protocol was used. 350 .mu.l of
RA1 buffer was used for cell lysis. Cells were frozen with RA1 at
-80.degree. C. for one day after which cells were thawed and the
lysate filtered for 1 min at 11,000 g. The filtrate was mixed with
350 .mu.l 70% ethanol in 1.5 ml tubes and centrifuged for 30 s at
11,000 g. 350 .mu.l of MDB buffer was added and the mixture
centrifuged for 1 min at 11,000 g. 95 .mu.l of Dnase reaction
mixture added and mixture left at room temperature for at least 15
min. Then wash with 200 .mu.l of RA2 buffer (to deactivate the
Dnase), and centrifuge for 30 s at 11,000 g. Wash with 600 .mu.l of
RA3 buffer, centrifuge for 30 second at 11,000 g. Wash with 250
.mu.l of RA3 buffer, centrifuge for 2 min at 11,000 g. Elute the
RNA with 60 .mu.l of Rnase-free H.sub.2O, centrifuge for 1 min at
11,000 g. Measure the concentration using Nanodrop (unit in
ng/.mu.l).
[0290] RT (reverse-transcription) experiments were performed as
follows. The following were mixed in a PCR tube: Random Primer (0.1
.mu.l), DNTP (1 .mu.l), RNA (250/500 ng), Rnase-Free H.sub.2O
(topped up to a final volume of 13 .mu.l). Incubate at 65.degree.
C. for 5 min. Incubate on ice for at least 1 min. Collect the
contents and centrifuge briefly before adding: 1.sup.st Strand
Buffer (4 .mu.l), DTT (1 .mu.l), RnaseOUT (1 .mu.l), SSIII Reverse
(1 .mu.l). Top up to final volume of 20 .mu.l. Mix by pipetting up
and down. Incubate at room temperature for 5 min. Incubate at
50.degree. C. for 60 mins. Inactivate the reaction at 70.degree. C.
for 15 min.
[0291] Reverse-transcription experiments were performed twice on
separate days and the PCR products pooled together and diluted to a
final concentration of 2.5 ng/.mu.l for subsequent Real-Time
PCR.
[0292] The Real-Time PCR was performed using a TaqMan.RTM. Fast
Universal PCR master Mix (2.times.) (Applied Biosystem). PCR master
Mix (10 .mu.l), ABI probe (1 .mu.l), cDNA (1 .mu.l), ddH.sub.2O (8
.mu.l). GAPDH and Beta actin were used as control genes against the
experimental targets OSX (osterix), OCN (Osteocalcin) and
Runx2.
Effect of BMP-2 Specific HS GAG+ on Osteogenesis
[0293] We evaluated the effects of the BMP-2 specific HS (GAG+)
isolated in Example 2 on osteogenesis by measuring the levels of
expression of osteogenic markers (osterix, Runx2, alkaline
phosphatase and BspII) by quantitative polymerase chain reaction
(qPCR). A time course was prepared to compare the expression of the
markers over a course of 10 days to compare the control to a low
and a high dose of BMP-2, the high dose being the optimal
conditions to induce differentiation of the cells.
Materials and Methods
[0294] Cells were seeded at 30,000 cell/cm.sup.2 in maintenance
media and left to attach overnight. The following day we switched
to differentiation media with: [0295] No additives [0296] 100 ng/ml
BMP-2 (positive control) [0297] 100 ng/ml BMP-2+30 .mu.g/ml-GAG
(Neg GAGs) [0298] 100 ng/ml BMP-2+30 .mu.g/ml+GAG (Pos GAGs) [0299]
100 ng/ml BMP-2+30 .mu.g/ml Heparin (Sigma # H3149) [0300] 100
ng/ml BMP-2+30 .mu.g/ml Total Heparan Sulfate (Sigma # H9902--HS
prior to fractionation)
[0301] The carbohydrates and BMP-2 were mixed together in the
smallest volume possible and incubated at room temperature for 30
minutes before their addition to the media and on the cells.
[0302] After 5 days, RNA was extracted using the Macherey-Nagel
kits and Reverse-Transcription was performed.
[0303] As we show in FIGS. 30-33, the Heparan sulfate from porcine
mucosa (Total HS) can increase the activity of BMP-2 (shown through
GAG+ induced increases in the expression of Alkaline Phosphatase,
osterix, BspII and Runx2) and this activity is contained within the
fraction that binds BMP2 (Pos GAGs). This means that we can isolate
the BMP enhancing fraction of a commercial HS by passing them on
the BMP-HBD peptide column.
Example 4
[0304] MC3T3-E1 (s14) preosteoblast cells (a mouse embryo calvaria
fibroblast cell line established from the calvaria of an embryo)
were expanded in .alpha.MEM media supplemented with 10% FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate and Penicillin/Streptomycin every
72 hours until sufficient cells were generated for plating. The
cells were differentiated by plating at 5.times.10.sup.4
cells/cm.sup.2 in .alpha.MEM media supplemented with 10% FCS, 2 mM
L-glutamine, 25 .mu.g/ml ascorbic acid, 10 mM .beta.-glycerol
phosphate and Penicillin/Streptomycin. The media was changed every
72 hours for 8 days at which point the cells and media were
harvested. The media was retained and clarified by high speed
centrifugation and filtration through a 0.4 .mu.m filter. The cell
layer was disrupted using a cell scraper and an extraction buffer
containing PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 1% CHAPS,
8 M Urea and 0.02% NaN.sub.3.
[0305] At all stages (unless otherwise stated), samples were
clarified before loading onto column systems. This process included
high speed centrifugation at 5000 g for 30 min, and filtration
through a 0.4 .mu.m syringe filter. The samples were always
clarified directly prior to loading through the column system to
prevent precipitates forming in stagnant solutions.
[0306] Anion exchange chromatography was used to isolate
proteoglycosaminoglycan (PGAG) fractions from both the media and
cell layer samples. In each case, the media or cell layer samples
were run through a Pharmacia XK 26 (56-1053-34) column packed with
Capto Q Anion Exchange Beads (Biorad) at a flow rate of 5 ml/min on
a Biologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector.
The samples were loaded in a low salt buffer containing PBS (150 mM
NaCl w/o Ca.sup.2+ and Mg.sup.2+), 100 mM NaCl, 0.02% NaN.sub.3 at
pH 7.3. The samples were eluted in a high salt buffer containing
PBS (150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and
0.02% NaN.sub.3 at pH 7.3. The relevant fractions were collected
and pooled into a single PGAG sample and lyophilized in preparation
for desalting.
[0307] The PGAG sample was desalted through four sequentially
joined Pharmacia HiPrep.TM.26/10 (17-5087-01) columns at a flow
rate of 10 ml/min on a Biologic DuoFlow system (Biorad) using a
QuadTec UV-Vis detector. The relevant fractions were collected and
pooled into a single sample set and lyophilized in preparation for
further treatment.
[0308] In the fourth step, the PGAG sample set obtained from the
desalting procedure was subjected to a pronase and neuraminidase
treatment, in order to digest away core proteins and to
subsequently liberate GAG chains. In this respect, lyophilized PGAG
samples were resuspended in a minimum volume of 25 mM sodium
acetate (pH 5.0) and clarified by filtration through a 0.4 .mu.m
syringe filter. The total sample volume was dispensed into 10 ml
glass tubes in 500 .mu.l aliquots. To this aliquot was added 500
.mu.l of 1 mg/ml neuraminidase before the mixture was incubated for
4 hours at 37.degree. C. Following incubation, 5 ml of 100 mM
Tris-acetate (pH 8.0) was added to each sample. An additional 1.2
ml of 10 mg/ml pronase, reconstituted in 500 mM Tris-acetate and 50
mM calcium acetate (pH 8.0), was added to each sample before the
mixture was incubated for 24 hrs at 36.degree. C. Following this
treatment, all volumes were combined and prepared for anion
exchange chromatography by centrifugation and filtration.
[0309] In a fifth step, the GAG sample isolated following protein
cleavage was eluted through a Pharmacia XK 26 (56-1053-34) column
packed with Capto Q Anion Exchange Beads (Biorad) at a flow rate of
5 ml/min on a Biologic DuoFlow system (Biorad) using a QuadTec
UV-Vis detector. In this respect, the sample was loaded in a low
salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and
Mg.sup.2+) and 0.02% NaN.sub.3 at pH 7.3. The sample was eluted in
a high salt buffer containing PBS (150 mM NaCl w/o Ca.sup.2+ and
Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH 7.3. The relevant
fractions were pooled, lyophilized and desalted as per the
aforementioned protocol for desalting the PGAG sample.
[0310] N-terminal biotinylated peptide (1 mg), corresponding to the
heparin-binding domain of BMP-2, and comprising an amino acid
sequence represented by QAKHKQRKRLKSSCKRH [SEQ ID NO: 17], was
mixed with low salt buffer containing PBS (150 mM NaCl w/o
Ca.sup.2+ and Mg.sup.2+). The mixture was eluted through a column
packed with a streptavidin-coated resin matrix. The column was then
exposed to a high salt buffer containing PBS (150 mM NaCl w/o
Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02% NaN.sub.3 at pH
7.3, to ascertain whether, under those conditions the peptide had
bound securely to the matrix. No substantial loss of peptide from
the column was observed. The column was subsequently washed with
the low salt buffer in preparation for sample loading.
[0311] The GAG mixture (2 mg), isolated using the procedure
outlined in Example 1, was suspended in low salt sodium phosphate
buffer (1 mL), and loaded onto the peptide column of Example 2. The
sample was eluted with a low salt buffer containing PBS (150 mM
NaCl w/o Ca.sup.2+ and Mg.sup.2+). A peak corresponding to GAGs
with negligible BMP-2 affinity was observed in the UV-Vis detector
trace. The column fractions responsible for giving rise to this
peak were combined. These fractions are known as `GAG-`--the minus
sign denoting the lack of affinity with the column. When it became
evident from the UV-Vis detector that the trace had flattened to
the baseline, and that no further oligosaccharide was eluting, the
eluting solvent was changed to a high salt buffer containing PBS
(150 mM NaCl w/o Ca.sup.2+ and Mg.sup.2+), 850 mM NaCl and 0.02%
NaN.sub.3 at pH 7.3. Following this change in the eluting solvent,
a peak corresponding to BMP-2 specific GAGs was observed in the
UV-Vis detector trace. The column fractions responsible for giving
rise to this peak were combined. These fractions are known as
`GAG+`--the plus sign denoting the presence of affinity with the
column. In the case of GAG compounds sourced from preosteoblast
cells, the GAG+ fraction represented 10% of the overall GAG
mixture.
Example 5
[0312] The addition of BMP2 has a clearly defined capacity to
induce osteogenic differentiation in C2C12 myoblasts. Similarly,
the pre-incubation of BMP2 with heparin has been shown to both
extend the cytokines half life and its immediate potency in vitro.
Here we examined the capacity of GAG+ and GAG- fractions to augment
the osteoinduction of C2C12 cells in vitro by BMP2.
[0313] The GAG+ sample from Example 4 (0, 10, 100, 1000 ng/mL) was
added to C2Cl2 myoblasts in vitro in the presence of BMP-2 (0, 50,
100 ng/mL). Measurement of the relative expression of the
osteocalcin gene indicated that the GAG+ sample was able to
potentiate BMP-2 to effect osteocalcin gene expression at levels of
BMP-2 far below those currently used in therapy (300 ng/mL). The
results of this assay (including calculated p-values and errors)
are represented graphically in FIG. 34 in which the experimental
conditions for each `culture condition` are as follows: [0314] 1.
Control cells, no BMP-2 added, no GAG added [0315] 2. BMP-2 at 50
ng/mL [0316] 3. BMP-2 at 50 ng/mL, GAG+ at 10 ng/mL [0317] 4. BMP-2
at 50 ng/mL, GAG+ at 100 ng/mL [0318] 5. BMP-2 at 50 ng/mL, GAG+ at
1000 ng/mL [0319] 6. BMP-2 at 100 ng/mL [0320] 7. BMP-2 at 100
ng/mL, GAG+ at 10 ng/mL [0321] 8. BMP-2 at 100 ng/mL, GAG+ at 100
ng/mL [0322] 9. BMP-2 at 100 ng/mL, GAG+ at 1000 ng/mL
[0323] Interestingly, while 1000 ng/ml of GAG+ is able to
significantly augment BMP2 mediated osteocalcin expression, the
addition of concentrations of GAG+ below 1000 ng/ml appear to
progressively inhibit this expression. Furthermore, the addition of
sufficient GAG+ also managed to drive the induction of osteocalcin
by 50 ng/ml of BMP2 above that of 100 ng/ml of BMP2 on its own,
indicating the potency of this interaction.
[0324] This cell culture based analysis demonstrated that the
addition of GAG+ to C2C12 osteogenic cultures together with BMP2
resulted in a significant upregulation of osteocalcin expression
indicating an increase in BMP2 signalling efficacy. This result
supports the specific association of GAG+ chains with BMP2, thereby
blocking the BMP2-HBP and preventing its association with
matrix-based PGAGs. The resulting upregulation of osteogenic gene
expression is comparable to that observed in previous studies
utilising heparin to achieve a similar effect. Interestingly, the
addition of concentrations of GAG+ that fall below 1000 ng/ml
appear to have an initially antagonistic effect on BMP2
signalling.
[0325] One possible hypothesis to explain this observation revolves
around the capacity for a given number of GAG+ molecules to bind a
certain number of BMP2 molecules. Under conditions where no
exogenous GAG+ is added to the culture system the majority of BMP2
molecules will be able to associate with the ECM, thereby being
localised away from their cognate receptors and being unable to
immediately initiate signalling. Subsequent dissociation of BMP2
from the ECM, both spontaneously and by targeted enzymatic
alteration of their associated GAG chains, has the capacity to
induce long term BMP2 signalling. The addition of a large number of
GAG+ molecules to this system, as is the case in samples
supplemented with 1000 ng/ml of GAG+, permits the majority of BMP2
molecules to remain in solution where they are free to mediate
receptor dimerisation and induce downstream signalling. Both these
processes of cytokine/receptor interaction likely require
particular concentration thresholds in order maintain an efficient
level of signalling. Under culture conditions containing 50 ng/ml
of BMP2, the addition of low concentrations of GAG+ allows for a
portion of the available cytokine to remain soluble while the
remaining portion associates with the ECM. Under these conditions
only a small quantity of BMP2 remains soluble but, due to its low
concentration, becomes highly diffuse in the media leading to
negligible signalling. Similarly, due to a portion of the BMP2
remaining solubilised, a reduced quantity of BMP2 can be found in
the ECM, resulting in a decrease in signalling from BMP2 liberated
from the ECM by direct cellular activity. However, under culture
conditions containing 100 ng/ml of BMP2 the combined effects of
soluble and ECM based BMP2 are, with the addition of 100 ng/ml of
GAG+, sufficient to induce BMP2 signalling similar to control
levels. Without further study, however, the dynamics involved in
BMP2/GAG+ signalling remain unclear. Future studies utilising
surface plasmon resonance may help elucidate the efficiency of
BMP2/GAG+ interactions and may aid in clarifying these
observations.
Example 6
[0326] The enzyme heparanase 3 was used to cleave GAG+ and GAG-
sugar chains from Example 4 according to the following method. GAG+
and GAG- were each treated separately at a concentration of 4
mg/mL, with heparanase 3 (250 mU enzyme per 100 .mu.g
oligosaccharide) for 16 hours at 37.degree. C. Subsequently, the
mixture was heated for 5 minutes at 70.degree. C. to inactivate the
heparanase 3. The digested GAG+ and GAG- mixtures were each
subjected to the peptide column separately. The UV-Vis detector
trace of each chromatographic run indicated that the digested
material showed the same affinity for the column as the undigested
material.
Sequence CWU 1
1
17118PRTMus musculus 1Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys Arg1 5 10 15His Pro219PRTUnknownAmino acid sequence
of heparin-binding domain of BMP4 protein 2Ser Pro Lys His His Ser
Gln Arg Ala Arg Lys Lys Asn Lys Asn Cys1 5 10 15Arg Arg
His331PRTUnknownAmino acid sequence of heparin-binding domain of
FGF2 protein 3Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala
Leu Lys Arg1 5 10 15Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro
Gly Gln Lys 20 25 30416PRTUnknownAmino acid sequence of
heparin-binding domain of SHH protein 4Gly Lys Arg Arg His Pro Lys
Lys Leu Thr Pro Leu Ala Tyr Lys Gln1 5 10 15538PRTUnknownAmino acid
sequence of heparin-binding domain of VEGF 189 protein 5Lys Cys Glu
Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys1 5 10 15Ser Val
Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser 20 25 30Arg
Tyr Lys Ser Trp Ser 35624PRTUnknownAmino acid sequence of
heparin-binding domain of FGFR1 protein 6Ala Pro Tyr Trp Thr Ser
Pro Glu Lys Met Glu Lys Lys Leu His Ala1 5 10 15Val Pro Ala Ala Lys
Thr Val Lys 20734PRTUnknownAmino acid sequence of heparin-binding
domain of Vitronectin protein 7Arg Pro Ser Leu Ala Lys Lys Gln Arg
Phe Arg His Arg Asn Arg Lys1 5 10 15Gly Tyr Arg Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn Ser 20 25 30Arg Arg817PRTUnknownAmino
acid sequence of heparin-binding domain of PDGF B protein 8Arg Val
Arg Arg Pro Pro Lys Gly Lys His Arg Lys Phe Lys His Thr1 5 10
15His923PRTUnknownAmino acid sequence of heparin-binding domain of
HB-EGF protein 9His Gly Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys
Lys Arg Asp1 5 10 15Pro Cys Leu Arg Lys Tyr Lys
201024PRTUnknownAmino acid sequence of heparin-binding domain of
FGFR3 protein 10Ala Pro Tyr Trp Thr Arg Pro Glu Arg Met Asp Lys Lys
Leu Leu Ala1 5 10 15Val Pro Ala Ala Asn Thr Val Arg
201113PRTUnknownAmino acid sequence of heparin-binding domain of
Fibronectin protein 11Thr Leu Glu Asn Val Ser Pro Pro Arg Arg Ala
Arg Val1 5 101220PRTUnknownAmino acid sequence of heparin-binding
domain of Laminin protein 12Arg Tyr Val Val Leu Pro Arg Pro Val Cys
Phe Glu Lys Gly Met Asn1 5 10 15Tyr Thr Val Arg
201318PRTUnknownAmino acid sequence of heparin-binding domain of
N-CAM protein 13Ile Trp Lys His Lys Gly Arg Asp Val Ile Leu Lys Lys
Asp Val Arg1 5 10 15Phe Ile14114PRTMus musculus 14Gln Ala Lys His
Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg1 5 10 15His Pro Leu
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile 20 25 30Val Ala
Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro 35 40 45Phe
Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln 50 55
60Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val65
70 75 80Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn
Glu 85 90 95Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly
Cys Gly 100 105 110Cys Arg156PRTArtificial sequenceSynthetic
sequence Consensus sequence for polypeptide heparin-binding domain
15Xaa Xaa Xaa Xaa Xaa Xaa1 5168PRTArtificial sequenceSynthetic
sequence Consensus sequence for polypeptide heparin-binding domain
16Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 51717PRTMus musculus 17Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg1 5 10
15His
* * * * *