Note: Descriptions are shown in the official language in which they were submitted.
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W093/20202 PCT/US93/03171
Decorin Fraoments and Methods of
Inhibitina Cell Re~ulatorv Factors
::
This invention was made with ~upport of
government grants CA 30199, CA 42507 and CA 28896 from the
National Cancer In~titute. Therefore, the United States
government may have certain rights in the inventio~
FIE D OF T~E INVENTIOM
This invention relates to cell biology and more
specifically to the control of cell proliferation by
inhibiting cell r~gulatory factors.
BACK~OUND OF T~E INVENTION
Proteoglycans are protein~ that carry one or more
glycosaminoglycan chains. The kno~n proteoglycans carry
out a wide variety of functions and are found in a variety
of cellular locations. ~any proteoglycans are compc~e~ts
of extracellular matrix, where they participate in the
acsembly of cells and effect the attachment of cells to the
matrix.
Decorin, al~o known a~ PG-II or PG-40, is a ~mall
2G proteoglycan produced by fibrobla~ts~ Its core protein has
a molecular weight of about 40,09U daltons. The core has
been sequenced (Kru~ius and Ruoslahti, Proc. Natl. Acad.
Sci. USA 83:76B3 ~19863; Day et al. Biochem. J. 248:801
(1987), both of which are incorporated herein by reference)
and it is known to carry a single glyco~amlnoglycan chain
of a chondroitin sulfate/dermatan sulfate type (Pearson, et
al., J. Biol. Chem. 258:15101 (1983), which is incorporated
herein by reference). The only previously known function
for decorin is binding to type I and type II collagen and
its effect on the fibril formation by these collagens
tVo~el, et al., Biochem. J. 223:587 ~1984); Schmidt et al.,
J. Cell Biol. 104:1683, ~1987)). Two proteoglycans,
biglycan (Fisher et al., J. Biol~ Chem. 264:4571 (1989))
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WOg3/20202 PCT/U~93/03171
and fibromodulin, (Oldberg et al., EMBO J. 8:2601, (1989)
have core proteins the amino acid sequences of which are
closely related to that of decorin and they, together with
decorin, can be considered a protein f~mily. Each of their
~equences is characterized by the presence of a leucine-
rich repeat of about 24 amino acids. Several other
proteins contain similar repeats. Together all of these
proteins form a superfamily of proteins (Ruoslahti, Ann.
Rev. Cell Biol. 4:229, (1988); McFarland et al., Science
245:494 (1989)).
Transforming growth factor ~s (TGF~) are a
f~ily of multi-functional cell regulatory factors produced
in various forms by many types of cells (for review see
Sporn et al., J. Cell Biol. 105:1039, (1987)). Five
different TGF~ are known, but the fu~ctions of only two,
TGF~-1 and TGP~-2, have been characterized in any detail.
TGF~'s are the subject of U.S. Patent Nos. 4,863,899;
4,816,561; and 4,742,003 which are incorporated by
reference~ TGF~-1 and TGF~-2 are publicly available
through many commercial sources (e.g. R & D Systems, Inc.,
Minneapolis, MN). These two proteins have sLmilar functions
and will be here collectively referred to as TGF~. TGFB
binds to cell surface receptors posse~sed by esg~ntially
all types of cells, causing profound changes in them. In
some cells, TGF~ promotes cell proliferation, in others it
suppres~es proliferation. A marked effect of TGF~ is that
it promotes the production of extracellular matrix proteins
and their receptors by cells (for review see Keski-Oja et
al., J~ Cell Biochem 33:~5 (19~7); Massague, Cell 4~:437
(1987); Roberts and Sporn in ~Peptides Growth Factors and
Their Receptors~ (Springer-Verlag, Heidelberg (1989)).
While TGF~ has many essential cell regulatory
functions, improper TGF~ activity can be detrimental to an
organism. Since the growth of mesenchyme and proliferation
of mesenchymal cells is stimulated by TGF~, some tumor
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W093/20202 PCT/US93/03171
cells may use TGF~ as an autocrine growth factor.
Therefore, if the growth factor activity of TGFB could be
prevented, tumor growth could be controlled. In other ca~es
the inhibition of cell proliferation by TGFB may be
detrLmental, in that it may prevent healing of injured
tissues. The stimulation of extracellular matrix
production by TGF~ is important in situations such as wound
healing. However, in some cases the body takes this
response too far and an excessive accumulation of
extracellular matrix ensues. An example of excessive
accumulation of extracellular matrix is glomerulonephritis,
a di~ease with a detrimental involvement of TGF~.
Thus, a need exists to develop compounds that can
modulate the effects of cell regulatory factors such as
TGFB. The present invention satisfies this need and
provides related advantages.
SUMMARY OF T~E INVENTI~N
The present invention provides active fragments
of proteins having a cell regulatory factor binding domain.
The invention further provides a method of inhibiting an
activity of a cell regulatory factor comprising contacting
the cell regulatory factor with a purified polypeptide,
wherein the polypeptide comprises a cell regulatory factor
binding domain of a protein and wherein the protein is
characterized by a leucine-rich repeat of about 24 amino
acids. In a specific embodiment, the present invention
relates to the ability of decorin, a 40,000 dalton protein
that usually carries a glycosaminoglycan chain, and more
specifically to active fragments of decorin or a functional
equivalent of decorin to bind TGFB or other cell regulatory
factors. The invention also provides a novel cell
regulatory factor designated Morphology Restoring Factor,
~MRF). Also provided are methods of identifying, detecting
and purifying cell regulatory factors and proteins that
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bind and affect the activity of cell regulatory factors.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows expression of decorin cDNA
containing a mutation of the serine acceptor ~ite to
alanine. COS-1 cultures were transfected with cDNA coding
for wild-type decorin (lane 1), decorin in which the
serine-4 residue was replaced by an alanine (lane 2), or
decorin in which the serine-4 residue was replaced by a
threonine (lane 3)~ Immunoprecipitations were performed
with an anti-decorin antibody and medium which was labeled
with 35S-sulfate (A) or 3H-leucine (B). Lane 4 shows an
immunoprecipitate from mock transfected COS-l cultures.
Arrow indicates top of gel. The numbers indicate Mr X 1 o-3
for molecular weight standards.
lS Figure 2 shows binding of [12sI~TGF~1 to decorin-
Sepharose: (A) Fractionation of [l25I]-TGF~1 by decorin-
Sepharose aff~nity chromatography~ r'2sI]TGF~l (5 x 105 cpm)
was incu~ated in BSA-coated polypropylene tubes with 0.2 ml
of packed decorin-Sepharose (~) or gelatin-Sepharose (o) in
2 ml of PBS p~ 7.4, containing 1 M ~aCl and 0.05% Tween 20.
After overnight incubation, the affinity matrices were
transferred into BSA-coated di~posable columns (Bio Rad)
and washed with the bindi~g ~uffer~ Elution was effected
first with 3 M NaCl in the binding buffer and then with 8
M urea in the s~me buffer: (B) Analysis of eluents of
decorin-Sepharose affinity chromatography by SDS-
polyacrylamide gel under nonreducing conditions. Lane 1:
the original [l25I3-labeled TGF~1 sample; lanes 2-7: flow
through and wash fractions; lanes 8-10: 3 M NaCl
fractions; lanes 11-14: 8 M urea fractions. Arrows .
indicate the top and bottom of the 12% separating gel. -
FigurP 3 shows the inhibition of binding of
[~2sI~TGF~31 to decorin by proteoglycans and their core
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W O 93/20202 P(~r/US93/03171
proteins: (A) Competition of [l25I]TGF~l binding to decorin-
coated microtiter wells by recombinant decorin (~), decorin
isolated from bovine ~kin (PGII) (~), biglycan isolated
from bovine articular cartilage (PGI) (-), chicken
S cartilage proteoglycan (o), and BSA (0). Each point
represents the mean of duplicate determinants. (B)
Competition of ['2sI]TGF~l binding with chondroitinase A~C-
treated proteoglycans and BSA. The concentrations of
competitors were expressed as intact proteoglycan. The
~ymbols are the same as in Figure 3A.
Figure 4 shows neutralization of the growth
regulating activity of TGF~1 by decorin: (A) Shows
inhibition of TGF~l-induced proliferation of CHO cells by
decorin. t3H]Thymidine incorporation assay was performed in
the presence of 5 ng/ml of TGF~-l and the indicated
concentrations of purified decorin (~) or BSA (o). At the
concentration used, TGF~-l induced a 50% increase of
t3~]thymidine incorporation in the CHO cells. The data
repre~ent percent neutralizatîon of this growth
stimulation; i.e. [3H]thymidine incorporation in the absence
of either TGF~l or decorin = 0~, incorporation in the
presence of TGF~ but not decorin = 109%. Each point shows
the mean ~ standard deviation of triplicate s2mples. (B~
Shows neutralization of TGF~1-induced growth inhibition in
MvlLu cells by decorin. The assay was performed as in A
except that TGF~-1 was added at 0.5 ng/ml. This
concentration of TGF~-1 induces 50~ reduction of
[3H]thym1dine incorporation in the Mvl~u cells. The data
represent neutralization of TGF~-induced growth inhibition;
i.e. [3H]thymidine incorporation in the presence of neither
TGF~ or decorin = 100%; incorporation in the presence of
TGF~ but not decorin = 0%.
Figure SA shows separation of growth inhibitory
activity from decorin-expressing CHO cells by gel
filtration. Serum-free conditioned medium of decorin
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W093/20202 - PCT/US93/03171
overexpre~or cëlls was fractionated by DEAE-Sepharose
chromatography in a neutral Tris-HCl buffer and fractions
containing growth inhibitory activity were pooled, made 4M
with guanidine-HCl and fractionated on a Sepharose CL-6
column equilibrated with the ~ame guanidine-HCl solution.
The fraction~ were analyzed for protein content, decorin
content, and growth regulatory activities. Elution
positions of marker proteins are indicated by arrows. BSA:
bovine ~erum albumin (Mr=66,000); CA: carbonic anhydrase
(Mr=29,000); Cy:cytochrome c (Mr=12,400); Ap:aprotinin
(Mr=6,500); TGF: ['25I]TGF~l (Mr=25,000).
Figure 5B shows identification of the growth
stimulatory material from gel filtration as TGF~l. The
growth stimulatory activity from the late fractions from
Sepharose 6B (bar in panel A) was ider~tified by inhibiting
the act~vity with protein A-purified ~gG from an anti-TGF~
anti~erum. Data represent percent inhibition of growth
~timulatory activity in a [3H]thymidine incorporation assay.
Each point shows the mean +standard deviation of triplicate
determinations. Anti-TGF~ , normal rabbit IgG (o).
Figure 6 is a schematic diagram of M9P-decorin
fragment fusion proteins. LRR is a leucine rich repeat.
MBP is maltose binding protein.
Figure 7 shows the results of binding studies of
l2sI-TGF~ to Lmmobilized recombinant decorin (DCl3) and MBP-
decorin fragments PT-65, PT-71, PT-72 and PT-73.
Figure 8 shows the results of binding studies of
I-TGF~ to immobilized decorin (DC-18v) and MBP-decorin
fragments PT-7l, PT-72, PT-84, PT-85, PT-86 and PT-87.
Figuré 9 shows the results of binding studies of
I-TGF~l to HepG2 cells in the presence of decorin
fragments PT-65, PT-71, PT-72 and PT-78.
W093/20202 2 1 3 2 8 ~ 9 PCT/US93/03171
Figure 10 shows the results of binding studies of
l2sI-TGF~ to ~-M(tk-) cells in the presence of decorin and
decorin fragments PT-71, PT-72, PT-84 and PT-85.
Figure 11 shows the results of binding studies of
~25I-TGF~l to ~-M(tk-) cells in the pre~ence of decorin and
recom~inant decorin fragments PT-71, PT-72, PT-86 and PT-
87.
Figure 12 shows the results of binding studies of
l25I-TGF~l to L-M(tk-) cells in the presence of synthetic
decorin peptide fragments P25-Q36, ~3l-S3, and H3l L42 and a
control peptide corresponding to the N-terminal 15 mer.
Figure 13 shows the re~ults of 125I-TGF-~ binding
to immobilized decorin with or without the presence of
synthetic decorin peptide ~ragments 16r" 16E, 16G and 16H
as well as a control peptide corresponding to the N-
terminal 15-mRr.
~ .
DETAILED DESCRlPTION OF T~E INVXNTION
The invention provides a method of inhibit~ng an
act.ivi~y of a cell regulatory factor comprising contacting
the cell regulatory factor with a purified polypeptide,
wherein ~he polypeptide comprises the cell regulatory
factor bi~ding domain of a protei~. The protein can be
characterized by a leucine-rich repeat of about 24 amino
acid~. Since di~eases such as cancer result from
uncontrolled cell proliferation, the invention can be used
to treat such diseases.
~ y "cell regulatory factor~' is meant a molecule
which can regulate an activity of a cell. The cell
regulatory factors are generally proteins which bind cell
surface receptors and include gr th factors. Examples of
cell regulatory factors inclllde the flve TGFB's, platelet-
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WO n/20202 PCT/US93/03171
derived growth factor (PDGF), epidermal growth factor,in~ulin like growth factor I and II, fibrobla~t growth
factor, interleukin-2, nerve growth factor, hemopoietic
cell growth factors (IL-3, GM-CSF, M-CSF, G-CSF,
S erythropoietin) and the newly discovered Morphology
Re~toring Factor, hereinafter ~MRF". Different regulatory
factors can be bound by different proteins which can affect
the regulatory factor~s activity. For example, TGF~-l is
bound by decorin and biglycan, and MRF by decorin.
By Ucell regulatory factor binding domain~ is
meant a fragment of a protein which binds to the cell
regulatory factor. A protein fragment that retains the
binding activity is included within the scope of the
invention and is referred to herein as an active fragment.
Fragments that retain ~uch activity, such as active
fragment~ of decorin or biglycan, can be recognized by
their ability to competitively inhibit the binding of, for
example, decorin to TGF~, or of other polypeptides to their
cognate growth factorc.
Active fragments can be obtained by proteolytic
digestion of the native polypeptide according to methods
known in the art or as described, for example, in Example
V~II. Alternatively, active fragments can be synthesized
based on the ~nown amino acid sequence by methods known to
2~ those ~killed in the art or as described in Example VIII.
The fragments can also be produced recombinantly by methods
known in the art or as described in Example V. Examples of
active fragments are included in Tables 4-15.
Such fragments can then be used in a competitive
assay to determine whether they retain binding activity.
For example, decorin can be attached to an affinity matrix,
as by the method of Example II. ~abelled TGFB and an
active fragment can then be contacted with the affinity
matrix and the amount of TGFB bound thereto determined.
` ' W0~3/~0202 2 1 3 2 8 S ~ PCT/Usg3/~3171
As used herein, "decorin" refers to a
proteoglycan having ~ubstantially the structural
characteristics attributed to it in Krusius and Ruoslahti,
supra . ~uman fibroblast decorin has substantially the
S amino acid sequence pre~ented in Krusius and Ruoslahti,
supra. "Decorin" refers both to the native composition and
to modifications thereof which substantially retain the
functional characteristics. Decorin core protein refers to
decorin that no longer is ~ubstantially substituted with
glycosaminoglycan and is included in the definition of
decorin. Decorin can be rendered glycosaminoglycan-free by
mutation or other means, such as by producing recombinant
decorin in cells incapable of attaching glycosaminoglycan
chains to a core protein.
lS Functional equivalents of decorin include
modificatioils of decorin that retain its functional
characteristics and molecules that are homologous to
decorin, such as the decorin family members biglycan and
fibromodulin, for example, that have the similar functional
activity of decorin. Modifications can include, for
example, the addition of one or more side chains that do
not interfere with the functional activity of the decorin
core protein.
Since the regulatory factor binding proteins each
contain leucine-rich repeats of about 24 amino acids which
can constitute 80~ of the protein, it is likely that the
fragments which retain the binding activity occur in the
leucine-rich repeats. However, it is possible the bi~ding
activity resides elsewhere such as in the carboxy terminal
amino acids or the junction of the repeats and the carboxy
terminal amino acids.
The invention teaches a general method whereby
one skilled in the art can identify proteins that can bind
to cell regulatory factors or identify cell resulatory
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' ' 10
factors that bind to a certain family of proteins. The
invention also teaches a general method in which these
novel proteins or known existing proteins can be assayed to
determine if they affect an activity of a cell regulatory
factor. Specifically, the invention teaches the discovery
that decorin and biglycan bind TGF~-l and MRF and that such
binding can inhibit the cell regulatory functions of TGF~-
1. Further, both decorin and biglycan are about 80%
homologous and contain a leucine-rich repeat of about 24
amino acids in which the arrangement of the leucine
residues is conserved. As defined, each repeat generally
contains at least two leucine residues and can contain five
or more. These prot~oglycans are thus considered members
of the same protein family. See Ruoslahti, supra, Fisher
et al., J. Biol. Chem., 264:4571-4576 (1989) and Patthy, J.
Mol. Biol., 198:567-577 (1987), all of which are
incorporated by reference. Other known or later discovered
proteins having this leucine-rich repeat, i.e.,
fibromodulin, would be expected to have a similar cell
regulatory activity. The ability of such proteins to bind
cell regulatory factors could easily be tested, for example
by affinity chromatography or microtiter assay as set forth
in Example II, using known cell regulatory factors, such as
TGFB-1. Alternatively, any later discovered cell
regulatory factor could be tested~ for example by affinity
chromatography using one or more regulatory factor binding
proteins. Once it is determined that such binding occurs,
the effect of the binding on the activity of all regulatory
factors can be determined by methods such as growth assays
as set forth in Example III. Moreover, one skilled in the
art could simply substitute a novel cell regulatory factor
for TGF~ 1 or a novel leucine-rich repeat protein for
decorin or biglycan in the Examples to determine their
activities. Thus, the invention provides general methods
to identify and test novel cell regulatory factors and
proteins which affect the activity of these factors.
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11
The invention also provides a novel purified
compound comprising a cell regulatory factor attached to a
purified polypeptide wherein the polypeptide comprise~ the
cell regulatory factor binding domain of a protein and the
protein is characterized by a leucine-rich repeat of about
24 amino acids.
The invention further provides a novel purified
protein, designated MRF, having a molecular weight of about
20 kd, which can be isolated from CH0 cells, copurifies
with decorin under nondissociating condition~, separates
from decorin under dissociating conditions, change~ the
morphology of transformed 3T3 cells, and has an activity
which is not inhibited with anti-TGF~-l antibody.
Additionally, MRF ~eparates from TGFB-l in ~PLC.
The invention still further provides a method of
purifying a cell regulatory factor comprising contacting
the regulatory factor with a protein which binds the cell
regulatory factor and has a leucine-rich repeat of about 24
amino acids and to purify the regulatory factor which
becomes bound to the protein. The method can be used, for
example, to purify TGF~-1 by using decorin.
The invention additionally provides a method of
treating a pathology caused by a TGFB-regulated activity
comprising contacting the TGF~ with a puri~ied polypeptide,
wherein the polypeptide comprises the TGF~ ~inding domain
of a protein and wherein the protein is characterized by a
leucine-rich repeat of about 24 amino acids, whereby the
pathology-causing activity is prevented or reduced. While
the method is generally applicable, specific examples of
pathologies which can be treated include a cancer, a
fibrotic disease, and glomerulonephritis. In cancer, for
example, decorin can be used to bind TGF~-1, destroying
TGF~-l's growth stimulating activity on the cancer cell.
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12
Finally, a method of preventing the inhibition of
a cell regulatory factor is provided. The method comprises
contacting a protein which inhibits an activity of a cell
regulator factor with a molecule which inhibits the
activity of the protein. For example, decorin could be
bound by a molecule, such as an antibody, which prevents
decorin from binding TGF~-l, thus preventing decorin from
inhibiting the TGF~-l activity. Thus, the TGF~-l wound
healing activity could be promoted by binding TGF~
inhibitors.
It is understood that modifications which do not
substantially affect the activity of the various molecules
of this invention including TGFB, MRF, decorin, biglycan
and fibromodulin are also included within the definition of
tho~e molecules. It is al80 understood that the core
protein& of decorin, ~iglycan and fibromodulin are also
included within the definition of those molecules.
."~.
The followin~ examples are intended to illustrate
but not limit the invention. -~
W093/20202 2 1 3 2 8 ~ 9 PCT/US93/03171
13
EXAMPLE I
EXPRESSION AND PURIFICATION OF RECOMBINANT DECORIN
AND DECORIN CORE PROTEIN
Expression System
The 1.8 kb full-length decorin cDNA described in
Krusi-s and Ruoslahti, Proc. Natl. Acad. Sci. USA 83:7683
(198~ ) which is incorporated herein by reference, was used
for the construction of decorin expression vectors. For
the expression of decorin core protein, cDNA was
mutagenized so the fourth codon, TCT, coding for ~;erine,
was changed to ACT codi~g for threonine, or GCT coding for
alanine. This was engineered by site-directed mutagenesis
according to the method of Kunkel, Proc. Natl. Acad. Sci
USA 82:488 (1985), which is incorporated herein by
reference. The presence of the appropriate mutation was
verified by DNA sequencing.
The m~mm~lian expression vectors pSV2-decorin and
pSV2-decorin/CP-thr4 core protein were constructed by
ligating the decorin cDNA or the mutagenized decorin cDNA
into 3.4 kb HindIII-Bam HI fragment of pSV2 (Mulligan and
~erg, Science 209:l423 (1980), which is incorporated herein
by reference). -
Dihydrofolate reductase (dhfr)-negative CHO cells
(C~O-DG44~ were cotransfected with pSV2-decorin or pSV2-
decorin/CP and pSV2dhfr by the calcium phosphatecoprecipitation method. The C~O DG44 cells transfected
with pSV2-decorin are deposited with the American Type
Culture Collection under Accession Number ATCC No. CRL
10~32. The transfected cells were cultured in nucleoside-
minus alpha-modified minimal essential medium (~-MEM),
(~IBCO, Long Island~ supplemented with 9% dialyzed fetal
calf serum, 2 mM glutamine, l00 units/ml penicillin and l00
~g/ml streptomycin. Colonies ar- ng from transfected
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14
cells were picked using cloning cylinders, expanded and
checked for the expression of decorin by
Lmmunoprecipitation from 35SO4-labeled culture ~upernatants.
Clones expressing a substantial amount of decorin were then
subjected to gene amplification by stepwi~e increasing
concentration of methotrexate (MTX) up to 0.64 ~M (Kaufman
and Sharp, J. Mol. Biol. 159:601 (1982), which is
incorporated herein by reference). All the amplified cell
lines were cloned either by lLmiting dilution or by picking
single MTX resistant colonies. Stock cultures of these
established cell lines were kept in MTX-containing medium.
Before use in protein production, cells were subcultured in
MTX-minus medium from stock cultures and passed at least
once in this medium to elLminate the possible MTX ef~Eects.
Alternatively, the core protein was expressed in
COS-l cells as dsscribed in Adams and Rose, Cell 41:1007,
(1985), which is incorporated herein by reference.
Briefly, 6-well multiwell plates were seeded with 3-5xl05
cells per 9.6 cm2 growth area and allowed to attach and grow
for 24 hour~. Cultures were transfected with plasmid DNA
when they were 50-70% confluent. Cell layers were washed
briefly with Tris buffered saline ~TBS) containing 50 ~M
Tris, l50 mM NaCl pH 7.2, supplemented with 1 mM CaCl2 and
0.5 mM MgCl2 at 37C to prevent detachment. The wells were
incubated for 30 minutes at 37C with 1 ml of the above
solution containing ? ~g of closed circular plasmid DNA and
0.5 mg/ml DEAE-Dextran (Sigma) of average molecular mass of
500,000. As a control, cultures were transfected with the
pSV2 expression plasmid lacking any decorin insert or mock
transfec~ed with no DNA. Culture were then incubated for
3 hours at 37C with Dulbecco's Modified Eayle's medium
(Irvine Scientific) containing 10% fetal calf serum and l00
yM chloroquine (Sigma), after removing the DNA/TBS/DEAE-
~extran solution and rinsing the wells with TBS. The cell
3~ layers were then rinsed twice and cultured in the above
medium, lacking any chloroquine, for approximately 36
WOg3/20202 213 2 8 5 9 PCT/US93/03171
hours. WI38 human embryonic lung fibroblasts were
routinely cultured in the same medium.
COS-l cultures were radiolabeled 36-48 hours
after transfection with the plasmid DNAs. All radiolabeled
S metabolic precursors were purchased from New England
Nuclear (Bosto~, MA). The isotopes used were 35S-sulfate
(460 mCi/ml), L-13,4,5-3H(N)] -leucine (140 Ci/ml) and L-
t~'C(U)] - amino acid mixture (product number 445E).
Cultures were labeled for 24 hours in Ham's F-12 medium
(GIBCO Labs), supplemented with l0~ dialyzed fetal calf
serum, 2 mM glutamine and 1 mM pyruvic acid, and containing
200 ~Ci/ml 35S-sulfate or 3H-leucine, or l0 yCi/ml of the
~C-amino acid mixture. The medium was collected,
~upplemented with 5 mM EDTA, 0.5 mM
phenylmethylsulfonylfluoride, 0.04 mg/ml aprotinin and l
~g/ml pepstatin to inhibit protease activity, fx~ed of
cellular debris by centrifugation for 20 minutes at 2,000
x G and stored at -20C. Cell extracts were prepared by
rin~ing the cell layers with TBS and then scraping with a
rubber policeman into 1 ml/well of ice cold cell lysis
buffer: 0.05 M Tris-HCl, 0.5 M NaCl, 0.1% BSA, 1% NP-40,
0O~% Triton X-l00, 0.l~ SDS, pH 8.3. The cell extracts
were clarified by centrifugation for 1.5 hours at 13,000 x
G at 4C.
Rabbit antiserum was prepared against a synthetic
peptide based on the first 15 residues of the mature form
of the human decorin core protein (Asp-Glu-Ala-Ser-Gly-Ile-
Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp). The synthetic
peptide and the antiserum against it have been described
elsewhere (Krusius and Ruoslahti, 1986 supra. ) Briefly,
the peptide was synthesized with a solid phase peptide
synthesizer (Applied Biosystems, Foster City, CA) by using
the chemistry suggested by the manufacturer. The peptide
was coupled to keyhole limpet hemocyanin by using N-
succinimidyl 3-(2-pyridyldithio) propionate (Pharmacia Fine
21328~9 ~...:;
W093/20202 PCT/US93/03171
16
Chemlcals, Piscataway, NJ) according to the manufacturer's
instructions. The resulting conjugates were emulsified in
Freund's complete adjuvant and injected into rabbits.
Further injections of conjugate in Freund's incomplete
adjuvant were given after one, two and three months. The
dose of each injection was equivalent to 0.6 mg of peptide.
Blood was collected l0 day~ after the third and fourth
injection. The antisera were tested against the
glutaraldehyde-cross linked peptides and isolated decorin
in ELISA (Engvall, Meth. Enzymol. 70:419-439 (1980)), in
immunoprecipitation and Lmmunoblotting, and by staining
cells in Lmmunofluorescence, as is well known in the art.
Immunoprecipitations were performed by adding 20
yl of antiserum to the conditioned medium or cell extract
collected from duplicate wells and then mixing overnight at
4C. Immunocomplexes were isolated by incubations for 2
hours at 4C with 20 ~l of packed Protein A-agarose
(Sigma). The beads were washed with the cell lysis buffer,
with three tube changes, and then washed twice with
phosphate-buffered saline prior to boiling in gel
electrophoresis sample buffer containing 10%
mercaptoethanol. Immunoprecipitated proteins were
separated by SDS-PAGE in 7.~-20% gradient gels or 7.5% non-
gradient gels as is well known in the art. Fluorography
was performed by usi~g Enlightning (New England Nuclear)
with intensification screens. Typical exposure tLmes were
for 7-l0 days at -70C. Autoradiographs were scanned with
an LKB Ultroscan XL ~nhanced Laser Densitometer to compare
the relative intensities and mobilities of the proteogIycan
bands.
SDS-PAG~ analysis of cell extracts and culture
medium from COS-l cells transfected with the decorin-pSV2
construct and metabolically radiolabeled with 35S-sulfate
revealed a sulfated band that was not present in mock-
transfected cells. Immunoprecipitation with the antiserum
-- 213285~
W093/20202 PCT/US93/03171
17
rai~ed against a synthetic peptide derived from the decorin
core protein showed that the new band was decorin.
Expression of the construct mutated such that the
~erine residue which is normally substituted with a
glycosAminoglycan (serine-4) was replaced by a threonine
residue by SDS-PAGE revealed only about 10% of the level of
proteoglycan obtained with the wild-type construct. The
rest of the immunoreactive material migrated at the
position of free core protein.
The alanine-mutated-cDNA construct when expre~sed
and analyzed in a similar manner yielded only core protein
and no proteoglycan form of decorin. Figure 1 shows the
expression of decorin (lanes 1) and its threonine-4 (lanes
3) and alanine-4 (lanes 2) mutated core proteins expressed
in COS cell transfectants. 35SO8-labeled (A) and 3H-leucine
labeled (B) culture supernatants were Lmmunoprecipitated
with rabbit antipeptide antiserum prepared against the NH2-
terminus of human decorin.
Purification of Decorin nd Decorin Core Protein from Spent
Culture Media
Cells transfected with pSV2-decorin vector and
amplified as described above and in Yamaguchi and
Ruo~lahti, Nature 36:244-246 (1988J, which is incorporated
herein by reference, were grown to 90% confluence in eight
culture flasks (175 cm2) in nucleoside minus a-MEM
supplemented with 9% dialyzed fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin and 100 ~g/ml
streptomycin. At 90% confluence culture media was changed
to 25 ml per flask of nucleoside-free a-MEM supplemented
with 6~ dialyzed fetal calf serum which had been passed
through a DEAE Sepharose Fast Flow column (Pharmacia)
equilibrated with 0.25 M NaCl in 0.05 M phosphate buffer,
pH 7.4. Cells were cultured for 3 days, spent media was
2132~
W093/20202 PCT/US93/03171
18
collectet and, immediately made to 0.5 mM
phenylmethylsulfonyl fluoride, l yg/ml pepstatin, 0.04
mg/ml aprotinin and 5 mM EDTA.
Four hundred milliliters of the spent media were
first passed through gelatin-SepharoFie to remove
fibronectin and materials which would bind to Sepharose.
The flow-through fraction was then mixed with DEAE-
Sepharose pre-equilibrated in 50 mM Tris/HCl, pH 7.4, plus
0.2 M NaCl and batch absorbed overnight at 4 C with gentle
mixing. The slurry was poured into a l.6 x 24 cm column,
washed extensively with 50 mM Tris/~Cl, pH 7.4, containing
0.2 M NaCl and eluted with 0.2 M - 0.8 M linear gradient of
NaCl in 50 mM Tris/HCl, pH 7.4. Decorin concentration was
determined by competitive ELISA as described in Yamaguchi
and Ruoslahti, supra. The fractions containing decorin were
pooled and further fractionate~1 on a Sephadex gel
filtration column equilibrated with 8 M urea in the Tris-
HCl buffer. Fractions containing decorin were collected.
The core protein is purified from cloned cell
lines transfected with the pSV2-decorin/CP vector or the
vector containing the alanine-mutated cDNA and amplified as
described above. The~e cells are grown to confluency as
described above. At confluency the cell monolayer is
washed four tLmes with serum-free medium and incubated in
a MEM supplemented with 2 mM glutamlne for 2 hours. This
spent medium is discarded. Cells are then incubated with
a MEM supplemented with 2 mM glutamine for 24 hours and the
spent media are collected and Lmmediately made to 0.5 mM
phenylmethylsulfonyl fluoride, l ~g/ml pepstatin, 0.04
mg/ml aprotinin and 5 mM EDTA as serum-free spent media.
The spent media are first passed through gelatin-Sepharose
and the flow-through fraction is then batch-absorbed to CM-
Sepharose Fast Flow (Pharmacia Fine Chemicals, Piscataway,
NJ) preequilibrated in 50 mM Tris/HCl, pH 7.4 containing
O.l M NaCl. After overnight incubation at 4C, the slurr~
2132859
W O 93/20202 P(~r/US93/03171 ~.
19
is poured into a column, washed extensively with the
preequilibration buffer and eluted with O.lM - lM linear
gradient of NaCl in 50 mM Tris/HCl, pH 7.4. The fractions
containing decorin are pooled, dialyzed against 50 mM
NB~HCO3 and lyophilized. The lyophilized material is
di~solved in 50 mM Tris, pH 7.4, containing 8M urea and
applied to a Sephacryl S-200 column (1.5 X 110 cm).
Fractions containing decorin core proteins as revealed by
SDS-polyacrylamide electrophoresis are collected and
represent purified decorin core protein.
. E~ PLE II
BINDING OF TGF~ TO DECORIN
A. Affinity Chromatoqraphy of TGF~ on Decorin-Sepharose
Decorin and gelatin were coupled to cyanogen
bromide-activated Sepharose (Sigma) by using 1 mg of
protein per ml of Sepharose matrix according to the
manufacturer's instructions. Commercially obtained TGF~-1
(Calbiochem, La Jolla, CA) was ~2sI-labelled by the
chlor~ine T method (Frolik et al., J. Biol. Chem.
2~9:10995-11000 (1984)~ which is incorporated herein by
reference and the labeled TGF~ was separated from the
unreacted iodine by gel filtration on Sephadex G-25,
equilibrated with phosphate buffered saline (PBS~
containing 0.1% bovine serum albumin (BSA) (Figure 2).
[l25I}-TGF~1 (5 x 105 cpm) was incubated in BSA-coated
polypropylene tubes with 0.2 ml of packed decorin-Sepharose
(~) or gelatin-Sepharose (o) in 2 ml of PBS pH 7.4,
containing 1 M NaCl and 0.05~ Tween 20. After overnigh;
incubation, the affinity matrices were transferred into
BSA-coated disposable columns (Bio Rad) and washed with the
binding buffer. Elution was effected first with 3 M NaCl
in the binding ~uffer and then with 8 M urea in the same
buffer. Fractions were collected, counted for
radioactivity in a gamma counter and analyzed by SDS-PAGE
2132~9 - : ~ ;~
W O 93/20202 P ~ /US93/03171
under nonreducing condition using 12% gels.
Figu~e 2A show~ the radioactivity profile from
the two columns and the SDS-PAGE analysis of the fractions
is shown in Figure 2B. The TGF~-1 starting material
contains a major band at 25 kd. This band represents the
native TGF~-l dimer. In addition, there are numerous minor
bands in the preparation. About 20-30% of the
radioactivity binds to the decorin column and elutes with
8 M urea, whereas only about 2% of the radioactivity is
present in the urea-eluted fraction in the control
fractionation performed on gelatin-Sepharose (Figure 2A).
The decorin-Sepharose nonbound fraction contains all of the
minor components and some of the 25 kd TGFB-1, whereas the
bound, urea-eluted fraction contains only T~F~-1 (Figure
2B). These results show ~hat TGF~-1 binds specifically to
decorin, since among the various components present in the
original TGF~-l preparation, only TGF~-1 bound to the
decorin-Sepharo~e affinity matrix and since there wa~ very
little binding to the control gelatin-Sepharo~e affinity
matrix. The TGF~-1 that did not bind to the decorin-
Sepharose column may have been denatured by the iodination.
Evidence for this pos~i~ility was provided by affinity
chromatography of unlabeled TGF~-1 as described ~elow.
In a second experiment, unlabeled TGFB-1 180 ng
was fractionated on decorin-Sepharose as described above
for 12sI-TGFB.
TGF~-1 (180 ng) was incubated with decorin-
Sepharose or BSA-agarose (O.2 ml packed volume) in PBS (pH
7.4) containing 1% BSA. After overnight incubation at 4C,
the resins were washed with 15 ml of the buffer and eluted
first with 5 ml of 3 M NaCl in PBS then with 5 ml of PBS
containing 8 M urea. Aliquots of each pool were dialyzed
against culture medium without serum and assayed for the
inhibition of [~H]thymidine incorporation in MvlLu cells
- 21328~3
W093/20202 PCT/US93/03171
21
(Example III). The amounts of TGF~-l in each pool were
calculated from the standard curve of t3H]thymidine
incorporation obtained from a parallel experLment with
known concentration of TGFB-l. The results ~how that the
TGF~-l bound e~sentially quantitatively to the decorin
column, whereas there was little binding to the control
c~lumn (Table 1). The partial recovery of the TGF~-l
activity may be due to loss of TGF~-l in the dialy~es.
TABLE I
Decorin-Sepharose affinity chromatography of nonlabeled
TGFB-l monitored by growth inhibition assay in MvlLu cells.
TGF~-l (ng)
Elution Decorin-Seph~ro~e BS~-Sepharose
Fl~w through & wash 2.7 ( 2-3%) 82.0 (93.9~)
3 M NaCl 2.2 ( 1.8%) 1~3 ( 1.5%)
8 M Urea 116.0 (95.9~) 4.0 ~ 4.6%~
B. Bindinq of TGF~-l to Decorin in a Microtiter Assav:
Inhibition bv Core Protein and Biqlycan
The binding of TGFB-l to decorin was also
examined in a microtiter binding a~say. To pexform the
assayt the wells of a 96-well microtiter plate were coated
overnight with 2yg/ml of recombinant decorin in 0.l M
sodium carbonate ~uffer, pH 9.5. The wells were washed
with PBS containing 0.05% Tween (PBS/Tween) and samples
containing 5 x 104 cpm of ['2sI3-TGFB-l and various
concentrations of competitors in PBS/Tween were added to
each well. The plates were then incubated at 37C for 4
3G hours (at 4C overnight in experiments with chondroitinase
ABC-digested proteoglycans), washed with PBS/Tween and the
bound radioactivity was solubilized with 1% SDS in 0.2 M
NaOH. Total binding without competitors was about 4% under
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W093/20202 PCT/US93/03171
22
the conditions used. Nvnspecific binding, determined by
adding l00-fold molar excess of unlabeled TGF~-l over the
labeled TGFB-l to the incubation mixture, was about 13% of
total binding. This assay was also u&ed to study the
ability of other decorin preparations and related proteins
to compete with the interaction.
Completion of the decorin binding was examined
with the following proteins (Figure 3; symbols are
indicated in the section of BRIEF DESCRIPTION OF T~E
10 FI~U~ES): ;
Decorin isolated from bovine skin and b;;glycan
isolated from bovine articular cartilage (PGI and PGII,
obtained from Dr. Lawrence Rosenberg, Monteflore ~edical
Center, N o Y ~; and described in ~osenberg et al., J. Biol.
Chem. 2~0:6304-6313, (1985), incorporated by reference
herein), chicken cartilage proteoglycan (provided by Dr.
Paul Goetinck, La Jolla Cancer Research Foundatio~, La
Jolla, CA, and described in Goetinck, P.F., in T~E
GLYCOCONJUGATES, Vol. III, ~orwitz, M.I., Editor, pp. 197-
2l7, Academic Press, NY) r For the preparation of coreproteins, proteoglyca~s were digested with chondroitinase
AB~ (Seikagaku, Tokyo, Japan) by incubating 500 ~g of
proteoglycan with Q.8 units of chondroiti~ase ABC in 250 yl
of 0.l M Tris/Cl, pH 8~0, 30 mM sodium acetate, 2 mM PMSF,
l0 mM N-ethylmalelmide, l0 mM EDTA, and 0.36 mM pepstatin
for 1 hour at 37C. Recombinant decorin and decorin
isolat~d from bovine skin (PGII) inhibited the binding of
E 125I~-TGF~-l, as expected (Figure 3A). Biglycan isolated
from bovine articular cartilage was as effective an
inhibitor as decorin. Since chicken cartilage
proteoglycan, which carries many chondroitin sulfate
chains, did not show any inhibition, the effect of decorin
and biglycan is unlikely to be due to glycosaminoglycans.
Bovine serum albumin did not shown any inhibition. This
notion was further supported by competition experiments
213285S
W O 93/20202 PC~r/US93/03171
23
with the mutated decorin core protein (not shown) and
chondroitinase ABC-digested decorin and biglycan (Figure
3B). Each of th~se proteins was inhibitory, whereas
cartilage proteoglycan core protein was not. The decorin
and biglycan core proteins were somewhat more active than
the intact proteoglycans. Bovine serum albumin treated
with chondroitinase A~C did not ~hown any inhibition.
Additional binding experiments ~howed that [l2sI]-TGFB-1
bound to microtiter wells coated with biglycan or its
chondroitinase-treated core protein. These results show
that TGFB-l binds to the core protein of decorin and
biglycan and implicates the leucine-rich repeats these
proteins share as the potential binding sites.
EXAMPL~ III
ANALYSIS OF THE EFFECT OF DECORIN ON CELL PROLIFERATION
STIMULATED OR INHIBITED BY TGFB-l
The ability of decorin to modulate the activity
of TGF~-l was examined in [3~]thymidine in~orporation
as~ays. In one assay, an unamplified C~O cell line
transfected only with pSV2dhfr (control cell line A in
reference 1, called CHO ~ells here3 was used. The cells
were maintained in nucleoside-free alpha-modified minLmal
essential medium (a-MEM, GIBCO, Long Island, -NY)
supplemented with 9% dialyzed fetal calf serum (dFCS) and
[3H]thymidine incorporation was assayed as described
(Cheifetz et al., Cell 48~409-415 ~1987)). TGF~-l was
added to the CH0 rell cultures at S ng/ml. At this
concentration, it induced a 50% increase of [3H]thymidine
incorporation in these cells. Decorin or BSA was added to
the medium at different concentrations. The results are
shown in Figure 4A. The data represent percent
neutralization of the TGF~ induced growth stimulation,
i.e., [3H]thymidine incorporation, in the absence of either
TGF~-1 or decorin = 0%, incorpor~tion in the presence of
TGFB-1 but not decorin = 100~. Each point shows the mean
213285~
W093/20202 PCT/US93/03171''"' "
24
+ standard deviation of triplicate samples. Decorin (~
~SA (o).
Decorin neutralized the growth stimulatory
activity of TGFB-l with a half maximal activity at about 5
~g/ml. Moreover, additional decorin suppressed the [3H]-
thymidine incorporation below the level observed without ,
any added TGF~-l, demonstrating that decorin al80 inhibited
TGF~ made by the CHO cells themselves. Both the decorin-
expressor and control C~O cells produced an apparently
active TGF~ concentration of about 0.25 ng/ml concentration
into their conditioned media as determined by the
inhibition of growth of the mink lung epithelial cells.
(The assay could be performed without interference from the
decorin in the culture media because, as shown below, the
effect of TGF~ on the mink cells was not ~ub~tantially
inhibited at the dec~rin concentrations pre~ent in the
decorin-producer media.) -
,~,
~xperLments in MvLu mink lung epithelial cell~
(American Type Culture Collection CCL64) also revealed an
effect by decorin on the activity of TGFn-l. Figure 4B
shows that in these cells~ the growth of which is measured
by thymidine incorporation, had been suppre~se~ by TGF~
Assay was performed as in Figure 4A, except that TGF~ was -,
added at 0.5 ng/ml. This concentration of TGEB induces 50%
reduction of [3H]-thymidine incorporation in the MvlLu
cells. The data represent neutralization of T~F~-induced
growth inhibition; i.e., [3H]-thymidine incorporation in the
,presence of neither TG~ or decorin = lO0%; incorporation
in the presence of TGF~ but not decorin = 0%. '
EXAMPLE IV
NEW DECORIN-BINDING FACTOR THAT CONTROLS CELL SPREADING
AND SAT~RATION DENSITY
Analysis of the decorin contained in the
2132859
W O 93/20202 P(~r/US93/03171
overexpressor culture media not only uncovered the
activities of decorin de6cribed above, but also revealed
the presence of other decorin-a~ociated growth regulatory
activitie~. The overexpres~or media were found to contain
a TGF~-like growth inhibitory activity. This was shown by
gel filtration of the DEAE-i~olated decorin under
dissociating conditions. Serum-free conditioned medium of
decorin overexpressor CHO-DG44 cells tran~fected with
decorin cDNA was fractionated by DEAE-Sepharose
chromatography in a neutral Tris-HCl buffer and fractions
containing growth inhibitory activity dialyzed against 50
mM NH,HCO3, lyophilized and dissolved in 4 M with guanidine-
FCl in a ~odium acetate buffer, pH 5.9. The dissolved
material was fractionated on a 1.5 x 70 cm Sepharose CL-6B
column equilibrated with the same guanidine-~Cl solution.
The fractions were analyzed by SDS-PAGE, decorin ELISA and
cell growth a~says, all described above. Three protein
peaks were obtained. One contained high molecular weight
proteins such as fibronectin (m.w. 500,000) and no
detectable growth regulatory activities, the second was
decorin with the activities described under Example III and
the third was a low molecular wei~ht (10,000-30,000-dalton~
fraction that had a growth inhibitory activity in the mink
cell as~ay and stLmulated the growth of the CHO cells.
Figure 5 summ~rizes these results. Shown are the ability
of the gel filtration fractions to affect [3H]-thymidine
incorporation by the C~O cells and the concentration of
decorin as determined by enzyme Lmmunoassay. Shown also
(arrows) are the elution positions of molecular size
markers: BSA, bovine serum albumin (Mr=66,000); CA,
carbonic anhydrase (Mr=29,000); Cy, cytochrome c
(Mr=12,400); AP, aprotinin (Mr=6,500); TGF, ['2sI]TGF~-l
~Mkz25,000).
.~,,
The nature of the growth regulatory activity
detected in the low molecular weight fraction was examined
with an anti-TGF~-l antiserum. The antiserum was prepared
213~3a9 ~ : ~
W093/20202 PCT/US93/03171
26
against a synthetic peptide from residues 78-lO9 of the
human mature TGF~-l. Antisera raised by others against a
cyclic form of the same peptide, the terminal cy~teine
residues of which were disulfide-linked, have previously
been shown to inhibit the binding of TGF~-l to its
receptors (Flanders et al., Biochemistry 27:739-746 (1988),
incorporated by reference herein). The peptide was
synthesized in an Applied Biosystems solid phase peptide
~ynthesizer and purified by ~PLC. A rabbit was immunized
subcutaneously with 2 mg per injection of the peptide which
was mixed with 0.5 mg of methylated BSA (5igma, St. Louis,
MO) and emulsified in Freund's complete adjuvant. The
injections were generally given four weeks apart and the
rabbit was bled approximately one week after the second and
every successive injection. The antisera u~ed in this work
has a titer (50% binding) of l:6,000 in radioimmunoassay,
bound to TGFB-l in immunoblots.
This antiserum was capable of inhibiting the
activity of purified TGF~-l on the CHO cells. Moreover, hS
5hown in Figure 5, the antiserum also inhibited the growth
stimulatory activity of the low molecular weight fraction
as determined by the t3~]-thymidine incorporation assay on
the CHO cells. Increasing concentrations of an IgG
fraction prepared from the anti-TGFB-l antiserum suppressed
the stLmulatory effect of the low molecular weight fraction
in a concentration-dependent manner (0)- IgG from a
normal rabbit serum had no effect in the assay (o).
The above result identified the stLmulatory
factor in the low molecular weight fraction as TGF~
However, TGF~-l is not the only active compound in that
fraction. Despite the" restoration of thymidine
incorporation by the anti-TGF~-l antibody shown in Figure
5, the cells treated with the low molecular weight fraction
were morphologically different from the cells treated with
the control IgG or cells treated with antibody alone. This
-` 2132859 -
WOg3/20202 PCT/U~93/03171
27
effect was particularly clear when the antibody-treated,
low molecular weight fraction was added to cultures of H-
ras transformed NIH 3T3 ~elle (Der et al., Proc. Natl.
Acad. Sci. USA 79:3637-3640 (1982)). Cells treated with
the low molecular weight fraction and antibody appeared
more spread and contact inhibited than the control cells.
This result shows that the CHO cell-derived recombinant
decorin is associated with a cell regulatory factor, MRF,
distinct from the well characterized TGF~'s.
Additional evidence that the new factor is
distinct from TGF~-l came from ~PLC experiments. Further
~eparations of the low molecular weight from the Sepharose
CL-6B column was done on a Vydac C4 reverse phase column (1
x 25 cm, 5 ~m particle size, the Separations Group,
~e~peria, CA) in 0.1% trifluoroacetic acid. Bound proteins
were eluted with a gradient of acetonitrite (22-40%) and
the factions were assayed for growth-inhibitory activity in
the mink lung epithelial cells and MRF activity in ~-ras
3T3 cells. The result showed that the TGF~-1 activity
eluted at the beginning of the gradient, whereas the MRF
activity eluted toward the end of the gradient.
EXAMPLE V
CONSTRUCTION AND EXPRESSION OF MBP-DECORIN
FRAGMENT FUSION PROq~EINS
MBP-Decorin fragment fusion proteins of varying
lengths were engineered such that the Maltose Binding
Protein (MBP) was attached to the amino terminus of the
gene encoding mature decorin as shown in Figure 6. The
techniques incorporated for such construction are described
in F. M. Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons (1987) and Maniatis et al.,
~olecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory (lg82), which are incorporated herein by
reference.
2132~a9
W093/20202 PCT/US93/03171
28
The decorin-encoding DNA fragments were generated
by polymerase chain reaction (PCR), Scharf et al., Science
233:1076-1078 (1986), which is incorporated herein by
reference. The prLmers, synthetic oligonucleotides
S obtained from Geno~ys (Houston, Texas), incorporate an Eco
RI restriction site at the 5' end and an Xba I restriction
sit~ at the 3~ end of the PCR product. In some instances,
the prLmers also included a base change to code for a
different amlno acid. The primers used to generate
specific inserts are identified in Table 2, while the
prLmer sequences are identified in Table 3. The template
DNA was a large scale CsCl prep of pPG-40 described in
Krusius and Ruoslahti, Proc. N~tl. Acad. Sci. ~SA 83:7683-
7687 ( 1986 ), incorporated herein by reference. The DNA
amplification reaction was done in a thermal cycler
according to manufacturer's recommendations (Perkin-Elmer
Cetus; Norwalk, Conneticut) using the Vent~ DNA Polymerase
(New England Biolabs; Beverly, Massachussets). The
decorin-encoding DNA fraqments cycled 35-40 times at 94,
40, and 72C.
-.
The PCR products were analyzed by agarose gel
electrophoresis, Ausubel et al., supra , and Maniatis et
al., supra, to identify and determine the decorin-encoding
DNA fragments (see Table 2 under "Insert Size"). The-PCR
products less than 200 ba~e pairs (bp) in size were
purified by electrophoresis onto DEAE-cellulose paper,
Ausubel et al., supra,; the PCR products greater than 200
bp were purified by using Prep-A-Gene~ DNA Purification Kit
(Bio-Rad; Richmond, California~ according to manufacturer's
~0 instructions.
The decorin-encoding DNA fraqments (insert) were
ligated between the Eco RI and Xba I restriction sites of
the polylinker in the vector pMAL-p (Protein Fusion and
Purification Sytem; New England Biolabs). The ligation had
a total of 500 ng of DNA and the molar ratio of
2 1~285~
W093/20202 PCT/US93/0317t
29
insert:vector was 3:1. The ligations were then transformed
into Escherichia coli (E. coli) DH5a cells (Gibco BRL;
Gaither~burg, Maryland), genotype: F- ~80dlacZ~M15,
~(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17(r~~, nk~),
supB44 A-~ thi-l, gyrA96, relAl, or E. coli Sure~ cells
(Stratagene, La Jolla, California), genotype: el4-(mcrA),
~(mcrCB-hsdSMR-mrr~171, sbcC, recB, recJ, umuC::Tn5(kanr),
uvrC, supE44, lac, syrA96, relA1, thi-l, endAl lF'prQAB,
lacIqZ~M15, TnlO, (tetr)]. The ~80dlacZ~N15 marker of the E.
coli DH5a ~train provides a-complementation of the ~
galactosida~e gene from pMAL-p. Colonies containing pMAL-p
with the decorin-encoding DNA fragments were colorless on
plates containinq 5-Bromo-4-chloro-3-indolyl-~-D-
galactoside (X-gal) due to the interruption of the ~-
galato~idase gene. Host cells containing pMAL-p only
produces blue colonies.
.
Minipreps of colorless colonies were then made as
described ~n Ausubel et al., supra, and Maniatis et al.,
supr~. Plasmids encoding MBP-decorin fragment fusion
proteins PT-73, -74, -75, -77, and -78, were then
digested with restriction endonucleases Eco RI and Xba I
(both from Promega; Madison, Wisconsin) and the presence of
specific in~erts confirmed by agaro~e gel elec~rophore~is.
The other plasmids encoding fusion protein~, PT-72, -76; -
84, -85, -86, and -~7, had inserts confirmed by sequencing
using 5equena~e~ Version 2.0 DNA Sequencing Kit (U. S.
Biochemical; Cleveland, Ohio~ according to manufacturer's
instructions.
Test expression of MBP-Decorin fragment fusion
proteins were performed in the host bacterial strain (see
Table 2). An overnight culture of E. coli DH5~ or E. coli
Sure~ cells containing the MBP-decorin fragment fusion
protein plasmids were made by taking a stab of a frozen
stock and inoculating L-Broth, Ausubel et al., supra,
containing 100 ~g/ml ampicillin at 37C with rapid shaking.
2l32~
W093/20202 PCT/US93/03171
The following morning, l ml was used to inoculate lO ml of
prewarmed medium (L-Broth containing ampicillin). After 1
hour at 37C, lO0 ~l of O.l M IPTG were added per culture
and the induced cultures were allowed to incubate for an
additional 2-3 hours. The cells were ly~ed by resuspending
in PAGE Sample Buffer (~ovex Experimental Technology;
Encinitas, California) with 0.8~ ~-mercaptoethanol and
~hearing lO times with a l cc tuberculin ~yringe. The
sample was run on an 8-16% gradient SDS-PAGE gel ~Novex
Experimental Technology) and a Western Blot (Novex
ExperLmental Technology) was performed a~cording to
manufacturer's recommendations. The blot was developed,
Ausubel et al., supra , with Rabbit anti-PG40 serum (Telios
Pharmaceuticals, Inc.; La Jolla, California) to test for
PT-65, -73, -74, -75, -76, -77, and -78, and Rab~it anti-
MBP serum (made in-house) to test for P~-72, -84~ -85, -86,
and -87. The results are indicated in Table 2 under "MW."
Large scale CsCl preps, Ausubel, et al., supra,
and Maniatus, et al., supra, of the plasmids encoding MBP-
Decorin fragment fusion proteins PT-84, -85, -86, and -87
were made and used to transform E. coli. DH5a. Expression
of the fusion proteins was confirmed by doing a test
expression as described above.
Production batches of the fusion proteins were
prepared as follows. An overnight culture of E. coli DHS~
cells contsining the MBP-Decorin fragment fusion protein
plasmids was made by taking a stab of the frozen stock and
inoculating L-Broth containing lO0 ~g/ml ampicillin at 37C
with rapid shaking. From this culture, 5 ml were used to
inoculate a larger 50 ml overnight culture. The following
morning, 50 ml of the larger culture were added to 500 ml
of pre-warmed media. Typically 1-4 liters were prepared
for each batch. After l hour at 37C, 5 ml of O.l M IPTG
were added per flask and the induced cultures were allowed
to incubate for an additional 2-3 hours. The cells were
--i 2132859
W093/20202 PCT/US93/03171
31
harvested by centrifugation at 5,000 rpm for lO minutes at
10C using either a GSA or GS-3 rotor in an RCSB centrifuge
(DuPont Instruments; Wilmington, Delaware). The pellets
were resuspended in O.l volume of lysis buffer (50 mM Tris-
HCl, pH 7.4, 150 mM NaCl, O.l M PMSF, and 0.25 mg/mllysozyme) and incubated for 10-15 minutes on ice. The
suspension was freeze/thawed three times by repeated
cycling through a dry ice/ethanol bath and a room
temperature shaking water bath. The suspen6ion was sheared
by homogenization using a dounce homogenizer. ~he lysate
was pre-cleared by centrifugation at 12,000 rpm for 30
minute~ in a SA-600 rotor (DuPont). The cleared
~upernatant was decanted and saved. A final clarification
step was done by centrifuging for 30 minutes at 4C in an
RC-80 ultracentrifuge using an AH-629 rotor (DuPont). The
final cleared lysates were stored either at 4C or -20C
until ready to be purified.
Affinity purifications of the MBP-Decorin fusion
proteins were done using an amylose resin (New England
Biolabs). 9riefly, six to seven ml of resin were packed
into a 2.5 x lO cm glass column in MBP column buffer (lO mM
Tris-HCl, pH 8.4, 1 mM EDTA, 0.5 M NaCl). The resin was
pre-equilibrated with at least 3 column volumes of MBP
column buffer containing 0.25% Tween 20. Cleared lysate as
prepared above was diluted l part lysate to 1 part 2X MBP
column buffer containing 0.5% Tween 20 and added to the
column at a flow rate of ~0-lO0 ml/ hr. Typically, 100-150
ml of diluted lysate were passed over each column. Non-
specific material was removed by washing with at least 3
column volumes each of MBP column ~uffer containing 0.25%
Tween 20 and MBP column buffer. The purified MBP-Decorin
fragment fusion protein was eluted with 5 x 4 ml aliquots
of MBP column buffer containing lO mM maltose. Peak
fractions containing the fusion protein were pooled,
assayed to determine protein quantity (Bio-Rad Protein Kit;
Richmond, California or Pierce BCA Protein Kit; Rockford,
213~ ~S~
W093/20202 PCT/US93/03171`
32
Illinois), run on an 8-16% SDS-PAGE gel (Novex Experimental
Technology), and stained with Coomasie Blue (Novex
ExperLmental Technology) to check for purity. The results
of the fusion protein are in Table I under "MW". The
purified fusion protein was stored at 4C or -20C in
aliquots until ready to be tested for activity.
The pMAL-p vector also was engineered such that
a termination codon was incorporated between the Eco RI and
Xba I sites. During this process, the original Eco RI site
in the vector was destroyed and replaced at a position
downstream from a second Factor Xa cleavage site. The
second Factor Xa site was incorporated to facilitate
subsequent cleavage of the decorin fusion protein from the
MBP carrier. The construction involved annealing
complLmentary oligos (OT-98 and OT-99; sequences in Table
3) a~d ligating into pMAL-p at the Eco RI and Xba I sites,
Ausubel, et al., supra, and Maniatis, et al., supra. The
ligation (PT-71) was transformed into E. coli DH5a cells,
mini-preps were made from colorless colonies and the clones
were sequenced for insert. Expression of the protein
followed the same procedure as the MBP-Decorin fragment
fusion proteins above. The results are indicated in Table
2 under "MW,
W O 93/20202 2 1 3 2 8 ~ ~ PC~r/US93/03171
'''~'
: -;
TAE~LE 2
_
CLoNE 5' 3' INSERr HOST # A~ MW
PRIMER PRIMER SIZE EALl~]Rl5L OF (K~)
(bp) STR~ ~ ..
. . _ _ ..
Pl-65 Oq-83 OT'85 990 DH5a 330 76
Mature Wh~le Decorin (29ner) (38mer)
. _ _
PT'i2 Oq-83 Oq-102137 D~5a 46 45
N~ =inal: (29mer) (3~mer)
C m~tated to Y
Fq~73 Oq-83 CT'103 285 D~5a 95 50
N~ cm. to LRR 2 (29mer) (36ner)
PT'74 ~q-83 Cq-104 420 DH5a 140 55
N~ m. to IRR 4 (2gmer) (32mer)
.
Pq-75 Cq-83 05-105 561 DH5~ 187 60N~ cm. to LRR 6 (2gmer) (32mer)
_ _ . .
Pq-76 CT'83 05-106 705 ~Sa 235 65 :~
~ m. to LRR 8 (29ner) (3~nPr)
. _ .
Pl-77 Oq~83 CT~107 ~43 DS5a 281 70
N~ 3n. to IRR 10 (2gmer) (3.~mer~
. ~ . . _
PI-78 oq-83 C~'108 918 DB~a 306 73
N~Erm. to % C're~m. (29mer) (32mer)
_ _ _
Pq'84 CI-83 CT'118 137 Sure~ 46 45 -:
N~ ~cm: (29mer) (77~er)
C nutated to S
1-4 _ . . ~
~I'85 Oq-83 Cq'll9 137 Sure~ 46 45
N~lerm: (29mer) (65~er) . :~
2~ C~_ ~utated to S
_ 3 _
PT'~6 Cq-120 0l-85 165 Sure~ 54 46
C'rerminal ~29mer~ (38mer)
. . _ __
-87 Oq-121 Oq-85 165 Sure~ 54 46
C'Term: (29me~) (3~mer~
C1 Dnr;ated to S .
. _ . _ _ . , . .
PT'71 ~ . . _ DH5a _ 40 .
ME~P
-= - . - - - .
C1 = the first cysteine
Cl_4 = the f-rst thrcugh the fcurth cvsteine
C2 3 = the second and third cvsteine
SUBSTITUTE SHEET
2 1 ~ ~ ~j 3 ~
WO 93/20202 PC~`/US93/03171 `
34
TABLE 3
Gl~.G~A.TQG. . . 3 '
C~103 5'.. (3G.q~T.AG~.q~.A~3G.A~T.A~C.m.~CT.AP~.m.~T~G.. 3'
~104 s~.. GG.T~r.A~.q~.m.Tc~.cAc.l~T.~r~G~.Qc... 3~
~106 5 ~ . . .GG.I~l~.A~.l~.A~T.~IC.A~.Al;C.A~.GP~.GCT. . . 3 '
C~107 5 ' . . .GG.~CT.AG~.I~.TCC.A~.I~.AG~.GP~.A~.(~IT.G. . . 3 '
OT-120 5'.. ~G.G~.llC.~.A~.G~C.qlC.TGC.C~.a~T.o.3'
C~98 5 ' . . .A~.m.P~C.GP.G.GOE .A~.~.G~A.lTC.q~A.T., . 3 '
aI~99 5 ' . . .Cr.A~.l~.G~A.llC.A~.CCT.AOC.~C.G~.A. . . 3 '
~` 2132859 ` -
i ~ 1
W093/20202 PCT/USg3/03171
.
Tables 4-15 below provide the nucleotide and
corresponding amino acid sequences of the decorin fragment fusion
proteins prepared as described above. Each table also identifies
the Eco RI and Xba I ligation sites.
2132`~S~
WO 93/20202 36 PCI`/US93/03171
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WO g3/20202 37 2 1 3 2 8 5 9 PCI-/US93/03171
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WO 93/20202 38
PCI/USg3/03171
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W O 93/20202 39 PC~r/US93~03171
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WO 93/20202 40 PCl`~US93/03171 `
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W093/20202 PCT/US93/03171
EXAMPLE VI
BlNDING STUDIES OF 125I-TGF-~
TO IMMOBILIZED DECORIN AND FRAGMENTS
Immulon wells were coated with 0.5 yg/ml
S recombinant decorin at 50 yl/well. The wells were placed
in a 37C incubator overnight and thereafter washed 3 times
with 200 yl P~S t0.15 M NaCl) per well to remove unbound
decorin. TGF-~ labeled with l25I (400 pM, New England
Nuclear, Bolton-~unter Labeled) was pre-incubated with or
without competitors in 200 ~l PBS/0.05% Tween-20 for l hour
and 45 minutes at room temperature. Competitors included
recombinant human decorin preparations (DC-l3 and DC-18v),
decorin fragments, and MBP as a negative control. DC-13
and DC-18v are different preparations of recombinant human
decorin; PT-71 or MBP (malto~e-binding protein) is a
negative control; PT-65 i~ MBP-whole decorin; PT-72 is MBP-
decorin N-terminus; PT-73 is PT-72 + 2 LRR; PT-84 and PT-85
are cysteine to ~erine mutant of PT-72; PT-86 is decorin C
terminu~; PT-87 is cy~teine to ~erine mutant of PT-86.
Fifty ~l/well of the pre-incubated 125I-$GF-~
mixture or control were added and incubated o~ernight at
0C. Following the incubation, 50 ~l of the free TGF-~
æupernatants were transferred to labeled tubes. The plate
was washed 3 tLmes with 0~05% Tween-20 in PBS (200
25 ~1/w811). The wells were then transferred into tubes for
counting in a gamma counter. The results of the binding
studies with Lmmobilized decorin are summarized in Figures
7 and 8.
Recombinant human decorin (DC-13), MBP-whole
30 decorin (PT-65), MBP-decorin N-terminus (PT-72) and MBP-
decorin N-terminus ~ 2 leucine rich repeats (PT-73)
inhibited l25I-TGF-~ binding to immobilized decorin as shown
in Figure 7. MBP alone (PT-7l~-had no effect on 12sI-TGF-
~binding to immobilized decorin. These results demonstrate
2 132~s'~
W093/20202 PCT/US93/03171
56
that the N-termlnus of decorin is capable of binding TGF-
~in ~olution and preventing it from binding to immobilized
decorin. Thus, two portions of the molecule appear to
contain part of the binding site in decorin for TGF-~.
As shown in Figure 8, recombinant human decorin
(DC-18V) + M~P-decorin N-terminus (PT-72) inhibited 12sI-TGF-
binding to immobilized decorin. In addition, cysteine to
~erine mutants of PT-72, (C-24, C-28, C-30, C-37 to serine,
PT-84; C-28 and C-30 to serine, PT-85) did not inhibit l2sI-
TGF-~ binding to decorin. MBP-decorin C-terminus (PT-86)
and a cy~teine to serine mutant (PT-87) of PT-86 also
inhibited l25I-TGF-~ binding_to decorin. These results
demonstrate that the C-terminus of decorin is also capable
of binding TGF-~ and that the first cysteine residue in the
C-terminus i8 not required for TGF-~ bin~ing.
EXAMPLE VII
BINDING OF l25I-TGF-~ TO ~EPG2 CELLS
About 2.5 x lO' HepG2 cells or L-M(tk-) cells were
incub~ted with 250 pM[l25I]TGF-~ in the presence of
recombinant human decorin (DC-12), PT 71 ~MBP), decorin
fragments (PT-72, -73, -84, -85, -86 and -87) or anti-
TGF-~ antibodies for 2 hours at room temperature. Cells
were washed with washing buffer ~l28 mM NaCl, 5 mM XCl, 5
mM Mg2SO~, 1.2 mM CaCl2, 50 mM HEPES, p~ 7.5) four times
before determination of bound CPM. The results are
~ummarized in Figures 9, lO and ll.
Recombinant human decorin, MBP-whole decorin (PT-
65), MBP-decorin N-terminus (PT-72) and MBP-decorin N-
terminus + 2 leucine rich repeats ~PT-78) inhibited l'5I-TGF-
~ binding to ~epG2 cells. MBP alone (PT-71) had no effect
on 12sI-TGF-~ binding to HepG2. These results shown in
Figure 9 demonstrate that the N-terminus of decorin is
capable of preventing TGF-~ from binding to its receptor on
W093/20202 21 3 2 8 S 9 PCT/US93/03171
57
HepG2 cells.
As shown in Figure 10, recombinant hu~an decorin
a~d MBP-decorin N-terminus (P~-72) inhibited l25I-TGF-
~binding to L-N(tk-) cells. An anti-TGF-Bl antibody also
inhibited TGF-~ binding to these cell~. Cysteine *o ~erine
mutants of PT-72 (C 24, C-28, C-30, C-37 to serine, PT-8~-
C-28 and C-30 to serine, PT-85) did not inhibit ~2sI-TGF~
binding to L-M(tk-) cells.
Recombinant h-lm~n decorin, MBP-decorin N-terminus
lo (PT-72) and anti-TGF-~ antibodies inhibited '2sI-TGF-
~binding to L-M(tk-) cells. MBP-decorin C-terminus (PT-86)
and a cysteine to serine mutant (PT-87) of PT-86 also
inhibited ~25I-TGF-~ binding to L-M(tk-) cells. As shown in
Figure 11, these results demonstrate that the C-terminus of
decorin also i~ capable of i~hibiting TGF-~ binding to its
receptor, and that the first cysteine residue of the C-
terminu~ is not required for inhibition.
':"
EXAMPI~ VIII
SYNTE~ETIC PEPTIDES
The following peptides were synthesized and
tested for their ability to inhibit binding of TGFB 1 to L- -
M(tk-) cells:
TABLE 16
Name Sequence
' ~3~ - S37 ~LR W QS
P2s - Q36 PFRSQSHLRVVQ
H31 - ~42 HLR W QSSDLGL
- nP2s ~ Q36 PFRCQC~LRWQ
Peptide H3, - S37 corresponds to the same decorin sequence
between His-31 and Cys-37 except the Cysteine at position
37 is replaced with a serine. Peptide P2s ~ Q36 corresponds
W093~ ~322~ ~ PCT/US93/03171
58
to the sequence reported in Krusius and Ruoslahti, supra,
from position Pro-25 through Gln-36, except the native
Cysteine residues at positions 28 and 30 are each replaced
with a serine. Peptide H3, - L,2 alco correæponds to decorin
between ~is-3l and Leu-42 except the cysteine at position
37 is replaced with a serine. Peptide nP2s-Q36 corresponds
exactly with decorin from position Pro-25 through Gln-36.
The peptides were synthesized using the applied
Biosystems, Inc. Model 43OA or 43lA automatic peptide
synthesizer and the chemistry provided by the manufacturer.
The activity of the peptides was evaluated using
the L-M~tk-) TGF~l binding inhibition assay described in
Example VlI, except various concentrations of peptide were
incubated with the cells and TGF~l instead of decorin and
rec~mbinant decorin fragments. The negative control wa~ a
synthetic peptide corresponding to the first 15 ~mino acids
of decorin, which has the sequence DEASGIGPEVPDDRD.
Figure 12 provides the binding data for peptides
P2S - Q36 ~ ~31 - S37 ~ and H31 - L~2 and the control peptide.
All three test peptides inhibited binding of TGF~l to L-
M(tk-) cells. Peptide nP2s - Q36~ in which the native Cys
residues remain, also demonstrated inhibitory activity,
al~eit to a lesser extent. Table 16 lists the test
peptides in the order of decreasing inhibitory acti~ity,
i.e., peptide ~3l-S3, was found to show the highest
inhibitory activity.
Further binding studies were conducted with
soluble N-terminal decorin peptide fragments synthesized
and tested for their ability to inhibit TGF-~l binding to
immobilized decorin as described above. The N-terminal
peptide fragments are listed in Table 17.
2132$5~
W093/20202 PCT/US~3/03171
ss -.
TABLE 17
Peptide Sequence
16D VPDDRDFEPSL&
1 6E FEPSLGPVCPFR
16G HLR W QCSDLGL
16H CSDLGLDKVPKDLPPD
~he results of the binding studies are shown in -
Fig~re 13, which shows that the peptide 16G inhibited TGF-~
binding to i~mo~ilized decorin.
lo Although the invention has been described with
reference to the presently-preferred embodiments, it should
be understood that various modifications can be made
without departing from the spirit of the invention.
Accordingly, the invention is l;m;ted only by the following
claim~.
