The Activation Sequence of Thrombospondin-1 Interacts with
the Latency-associated Peptide to Regulate Activation of Latent
Transforming Growth Factor-
*
Solange M. F.
Ribeiro
§¶,
Maria
Poczatek
§
,
Stacey
Schultz-Cherry
**,
Matteo
Villain
, and
Joanne E.
Murphy-Ullrich
From the
Department of Pathology, Division of
Molecular and Cellular Pathology, and the

Department of Physiology and Biophysics,
University of Alabama at Birmingham,
Birmingham, Alabama 35294-0019
 |
ABSTRACT |
One of the primary points of regulation of
transforming growth factor-
(TGF-
) activity is control of its
conversion from the latent precursor to the biologically active form.
We have identified thrombospondin-1 as a major physiological regulator of latent TGF-
activation. Activation is dependent on the
interaction of a specific sequence in thrombospondin-1
(K412RFK415) with the latent TGF-
complex. Platelet thrombospon-din-1 has TGF-
activity and
immunoreactive mature TGF-
associated with it. We now report that
the latency-associated peptide (LAP) of the latent TGF-
complex also
interacts with thrombospondin-1 as part of a biologically active
complex. Thrombospondin·LAP complex formation involves the activation
sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the
amino terminus of LAP that is conserved in TGF-
1-5. The
interactions of LAP with thrombospondin-1 through the LSKL and KRFK
sequences are important for thrombospondin-mediated activation of
latent TGF-
since LSKL peptides can competitively inhibit latent
TGF-
activation by thrombospondin or KRFK-containing peptides. In
addition, the association of LAP with thrombospondin-1 may function to
prevent the re-formation of an inactive LAP·TGF-
complex since
thrombospondin-bound LAP no longer confers latency on active TGF-
.
The mechanism of TGF-
activation by thrombospondin-1 appears to be
conserved among TGF-
isoforms as latent TGF-
2 can
also be activated by thrombospondin-1 or KRFK peptides in a manner that
is sensitive to inhibition by LSKL peptides.
 |
INTRODUCTION |
Transforming growth factors-
are a family of small peptide
growth factors (25 kDa) involved in the regulation of a variety of
cellular functions (1-3). Processes regulated by
TGF-
1 include
angiogenesis, embryogenesis, wound healing, and inflammation. There are
five isoforms of TGF-
, three of which are expressed in mammals.
TGF-
is synthesized by virtually all cell types in a latent form
that must be activated in order to be recognized by cell-surface
receptors and to trigger biological responses (1-4). Mechanisms
controlling conversion of the latent complex to the active state are
key regulators of TGF-
activity (1-4). Physiological mechanisms of
activation are not well understood, although proteolytic processing by
plasmin, exposure to reactive oxygen species, and binding to integrins
may participate in this process (4, 47). Our laboratory has shown that
interaction of latent TGF-
with the multifunctional platelet and
matrix protein thrombospondin-1 (5-10) results in activation of latent
TGF-
(12-15). Thrombospondin purified from human platelets
(thrombospondin-1) is associated with TGF-
activity (11). The site
in thrombospondin responsible for latent TGF-
activation has been
localized to the type 1 repeats (14): specifically, the KRFK sequence
located between the first and second type 1 repeats of thrombospondin-1 (15). To better understand the mechanism of thrombospondin-mediated activation of latent TGF-
, we sought to determine the region of the
latent TGF-
complex recognized by the TGF-
-activating sequence
KRFK in thrombospondin.
Small latent TGF-
(reviewed in Refs. 1-4) is a dimeric complex of
~100 kDa, composed of two identical chains in which an amino-terminal
278-amino acid latency-associated peptide (LAP) is noncovalently
associated with the carboxyl-terminal 112-amino acid active peptide.
This latent complex is the product of a single gene. Prior to
secretion, LAP is enzymatically cleaved from the active peptide (16),
and the integrity and latency of the secreted complex are presumably
maintained via electrostatic interactions (17). Although latent TGF-
can also exist as a large complex in which small latent TGF-
is
associated with a latent TGF-
-binding protein, the presence of the
latent TGF-
-binding protein is neither necessary nor sufficient to
confer latency on the active peptide (18-20). On the other hand,
latency is dependent on the presence of LAP, and modification of the
cysteine residues responsible for LAP dimerization results in altered
TGF-
secretion (21-23), suggesting that the tertiary structure of
LAP is important for the formation of the latent TGF-
complex.
Gentry and co-workers (21) showed through mutagenesis studies that
amino acids 40-80 in LAP are important for maintenance of the latency
of the complex. In a previous study, we observed that antibodies raised
against a sequence present in the amino terminus of LAP (residues
81-94) inhibited activation of latent TGF-
by thrombospondin (13). These observations led us to propose that thrombospondin-mediated activation of latent TGF-
involves interactions between the
thrombospondin activation sequence (KRFK) and a site present in the
amino-terminal region of LAP.
We now show that LAP is complexed with thrombospondin-1 in association
with biologically active TGF-
and that the thrombospondin-1 sequence
KRFK binds LAP through interactions that involve a specific sequence at
the amino terminus of
1-LAP
(L54SKL57). The KRFK sequence in
thrombospondin-1 and the LSKL sequence in LAP are apparently critical
for activation of latent TGF-
by thrombospondin-1 since soluble LSKL
peptides can competitively block activation of latent TGF-
by either
thrombospondin-1 or KRFK-containing peptides. LAP binding to
thrombospondin may play a role in preventing re-formation of the latent
complex. In addition, the mechanism of thrombospondin-mediated
activation of latent TGF-
appears to be conserved in the mammalian
isoforms of TGF-
since thrombospondin-1 can also activate latent
TGF-
2 in an LSKL-sensitive manner.
 |
EXPERIMENTAL PROCEDURES |
Thrombospondin Purification--
Thrombospondin-1, native or
strip- ped of TGF-
activity, was purified as described (11) from
human platelets obtained from the Birmingham American Red Cross.
Thrombospondin purity was assessed by SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining. The depletion of TGF-
activity in preparations of stripped thrombospondin was confirmed in
the NRK colony formation assay (11).
Peptides, Antibodies, and Other Reagents--
The peptides used
in this work were synthesized by the University of Alabama at
Birmingham Comprehensive Cancer Center/Peptide Synthesis and Analysis
shared facility. Initial peptides and peptide 246 used in this study
were a gift from Dr. David Roberts (NCI, National Institutes of
Health). Recombinant latent TGF-
2 was a generous gift
from Dr. Patricia Segarini and Celtrix Corp. (Palo Alto, CA), and was
purified as described (24). Recombinant latent TGF-
1 was
a gift from Jane Ranchelis (Bristol-Myers Squibb, Seattle, WA).
Monoclonal antibody 133 against stripped thrombospondin-1 was developed
in a joint effort between our laboratory and the University of Alabama
at Birmingham Hybridoma core facility (12). Recombinant human
1-LAP (catalogue no. 246-LP/CF) and mouse monoclonal and
goat polyclonal anti-LAP antibodies (catalogue no. AB-246-NA) were
purchased from R&D Systems (Minneapolis, MN). Secondary antibodies were
purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Cells--
Bovine aortic endothelial (BAE) cells were isolated
in our laboratory from aortas obtained at a local abattoir and were
characterized by Dil-AcLDL uptake and staining for factor VIII antigen,
according to established protocols. Stocks were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter
glucose and supplemented with 20% fetal bovine serum. Conditioned
media experiments were performed in the presence of reduced serum
concentrations as described in the figure legends. NRK-49F cells
(CRL-1570) were purchased from the American Type Culture Collection
(Rockville, MD) and were kept in DMEM supplemented with 10% calf
serum. All cells were routinely tested for mycoplasma.
Activation of Latent TGF-
by Thrombospondin or
Peptides--
Equimolar concentrations of stripped thrombospondin-1 or
peptides (11 nM) were incubated with recombinant latent
TGF-
(2 nM) in a final volume of 0.5 ml of PBS for
1 h at 37 °C (13). Alternatively, stripped thrombospondin-1 or
peptides were incubated with BAE cell-conditioned media as described
(11). A positive control for latent TGF-
activation consisted of
heat treatment of the latent complex at 80 °C for 5 min.
Assay of TGF-
Activity--
TGF-
activity was assayed
based on its ability to stimulate growth of NRK fibroblasts in
suspension as described (11). In brief, 1-3 × 103
NRK cells were plated in a 0.3% agar suspension in the presence of
epidermal growth factor (2.5 ng/ml; Life Technologies, Inc.) and in the
presence or absence of TGF-
and incubated at 37 °C for 7 days. At
the end of this incubation period, colonies containing 8-10 cells
(i.e. colonies larger than 62 µm) were counted. Active TGF-
(2.5 ng/ml) was used as a positive control. Experiments were
performed in triplicate at least twice.
Western Blots--
Samples were separated by SDS-polyacrylamide
gel electrophoresis (% acrylamide indicated in the figure legends) and
transferred to nitrocellulose membranes (2 h, 100 V). Nonspecific
protein-binding sites present in the membranes were blocked by
incubation with 0.5% bovine serum albumin in Tris-buffered saline
containing 0.05% Tween 20 (Tris-buffered saline/Tween). Membranes were
then incubated with primary antibodies diluted in Tris-buffered
saline/Tween (antibody 133 used at 0.05 µg/ml, goat polyclonal
anti-LAP at 1 µg/ml, and other antibodies and dilutions specified in
the figure legends) followed by extensive washes in Tris-buffered
saline/Tween with 0.1% Tween 20. After washing, membranes were
incubated with peroxidase-conjugated secondary antibodies
(peroxidase-conjugated goat anti-mouse IgG used at 0.1 µg/ml for
1 h at room temperature, peroxidase-conjugated rabbit anti-goat
IgG at 0.08 µg/ml, and dilutions and incubation times for other
antibodies as indicated in the figure legends) and developed by
enhanced chemiluminescence (Pierce) according to the manufacturer's
instructions. Multiple exposures were obtained to assure linearity of
the response.
Peptide Affinity Column--
Peptide KRFKQDGGC or TRIRQDGGC (5 mg/1 ml in 50 mM Tris and 5 mM EDTA, pH 8.5)
was coupled to Sulfolink (1 ml; Pierce) according to the
manufacturer's instructions and equilibrated in PBS. 2.4 µmol of
peptide KRFKQDGGC or 3.3 µmol of peptide TRIRQDGGC were coupled to
the Sulfolink resin. Recombinant human
1-LAP (10 µg/0.5 ml of PBS, 0.28 nmol) was loaded and incubated with the
affinity matrix (bed volume = 1 ml) for 20 min at room temperature
and then circulated through the column five times. Prior to elution, the column was washed with 25 ml of PBS. Proteins bound to the affinity
matrix were then eluted stepwise, first with 4 ml of peptide SLLK,
followed by 10 ml of peptide LSKL and, for the TRIR affinity column,
peptide TRIR (all peptides at 86 µM, a 150-fold molar
excess to LAP). Fraction size was 0.25 ml, and all LAP protein eluted
between fractions 3 and 5 (0.75-1.25 ml). Eluted proteins were
separated by SDS-polyacrylamide gel electrophoresis and analyzed by
Western blotting with anti-LAP antibodies.
Immunoprecipitation--
Stripped thrombospondin (11 nM) and recombinant human
1-LAP (28 nM) were incubated together in a total volume of 0.5 ml of PBS in the presence or absence of peptide KRFK, TRIR, or KRAK, (11 µM) or peptide LSKL, SLLK, or RGQILSKLRL (28 µM). Peptides were used at a 1000-fold molar excess to
either TSP or LAP, respectively. When peptides were present, each
protein was preincubated with the appropriate inhibitory peptide for 30 min at 4 °C (TSP preincubated with LSKL and LAP preincubated with
KRFK). The second protein was then added to the peptide/protein mixture
and incubated together for 1 h at 4 °C. The protein mixture was
incubated for 1 h at 4 °C with goat polyclonal antibodies
raised against recombinant human LAP (0.5 µg of antibody/0.5 ml of
sample), followed by a 30-min incubation with protein G-Sepharose 4B
beads (Sigma) in 10 mM Tris, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, and 0.5% Nonidet P-40.
Alternatively, the protein mixture was incubated for 1 h at
4 °C with GammaBind G-Sepharose (catalogue no. 17-0885-01, Amersham
Pharmacia Biotech) conjugated with monoclonal antibody 133 in wash
buffer (PBS containing 1 g/liter ovalbumin and 5 ml/liter Tween 20; 10 µg of antibody/0.5 ml of matrix). Immune complexes were washed with
wash buffer, resuspended in reducing Laemmli buffer, and analyzed by
Western blotting with monoclonal antibody 133 against thrombospondin or
with goat polyclonal anti-LAP antibodies. For dose-response inhibition
assays, LAP was preincubated with 0.11-110 µM peptide
KRFK, whereas thrombospondin was incubated with 0.28-280
µM peptide LSKL.
For immunoprecipitation of proteins from media conditioned by BAE
cells, ~3 × 106 cells were incubated for 24 h
in 3 ml of DMEM containing insulin-transferrin-sodium selenite media
supplement (Sigma) at the concentration recommended by the
manufacturer. Conditioned media were harvested and immediately incubated with goat polyclonal antibodies against recombinant human LAP
(10 µg/ml) for 1 h at 4 °C, followed by incubation with protein G-Sepharose 4B beads for 1 h at 4 °C. Immune complexes were washed and analyzed by Western blotting with monoclonal anti-TSP antibodies as described above.
LAP Immunodepletion Experiments--
To deplete native
thrombospondin of LAP, 20 µg of thrombospondin in 25 µl of PBS were
incubated three times with 25 µl of goat anti-LAP antibodies coupled
to CNBr-activated Sepharose (coupling per manufacturer's instructions)
for 20 min each time. Following each incubation, samples were
centrifuged, and the supernatant was transferred to another tube
containing antibodies coupled to resin. Prior to the first incubation
and following the last incubation, protein concentration in the sample
was measured by A280 nm using a molar
extinction coefficient of 1.27. Sample volumes to be tested for TGF-
activity and Western-blotted for LAP were adjusted so that the same
amount of protein (6.25 µg) was used in all cases.
Analysis of TGF-
Activity in Complexes--
Assay conditions
were those previously described as ideal for re-formation of the latent
TGF-
complex (22). In brief, thrombospondin (9 µg; amount chosen
based on our previous studies of latent TGF-
activation by
thrombospondin) was incubated with LAP (28 ng) in 100 µl of
serum-free DMEM for 1 h at room temperature. TGF-
(2 ng in 2 µl) was then added to the appropriate samples, followed by an
additional incubation for 1 h at room temperature. Samples to
which no TGF-
was added, samples containing TGF-
alone, and samples in which LAP and thrombospondin were not incubated together prior to addition of TGF-
were incubated at the same temperature for
the same extent of time to minimize variations due to loss of protein
to the tube or loss of TGF-
activity over time. Immediately following the second incubation, samples were tested for TGF-
activity as described above.
Hydropathic Complementarity Analysis--
The search for a
sequence in LAP complementary to the thrombospondin-1 sequence KRFK was
performed through computer analysis utilizing a computer program
designed to identify patterns of inverted hydropathy (25). The
parameter settings used were as follows: 1) search a window size of
five amino acids (hits are searched for in a window of five residues,
sliding the window down the sequence one amino acid at a time); 2)
average chain complementarity set at 1.2 (this value represents the
average of the differences in the hydropathic scores of aligned amino acids for the window size selected; the closer to 0, the better the
complementarity); and 3) the cutoff point for considering if two amino
acids are opposite set to 2.0 (the absolute value of the two aligned
residues added together is denoted as the cutoff).
 |
RESULTS |
LAP Co-purifies with Thrombospondin-1 Secreted by Human Platelets
and by BAE Cells--
Previous results indicated that an antibody
raised against the amino terminus of LAP could block
thrombospondin-mediated activation of latent TGF-
(13), suggesting a
possible interaction of thrombospondin with the LAP portion of the
latent complex. During the course of our studies, Yang et
al. (45) reported that recombinant dimeric LAP binds to
immobilized thrombospondin. Since the presence of LAP is both necessary
and sufficient to confer latency on TGF-
and since TGF-
associated with thrombospondin-1 is in its active state, one would
predict that LAP would not be present in biologically active
thrombospondin-1·TGF-
complexes. However, human platelet thrombospondin-1 that has TGF-
bioactivity (11) also contains detectable LAP, suggesting that LAP may potentially be associated with
thrombospondin-1·TGF-
complexes (Fig.
1A). Furthermore, LAP isolated
by immunoprecipitation from media conditioned by BAE cells in culture
co-purifies with thrombospondin-1, as detected by Western blotting
(Fig. 1B). These observations show that in biological
fluids, thrombospondin and LAP can exist in complexes. Furthermore,
these data suggest the possibility that active TGF-
can form a
ternary complex with thrombospondin-1 and LAP.

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Fig. 1.
Native thrombospondin-1 contains associated
LAP. A, native TSP1 was subjected to SDS-polyacrylamide
gel electrophoresis on a 5-15% gradient gel under reducing
conditions, transferred to nitrocellulose membranes, and analyzed by
Western blotting (WB) with mouse monoclonal anti-LAP
antibodies (2 µg/ml, 4 h, room temperature). The secondary
antibody used was peroxidase-conjugated goat anti-mouse IgG (0.1 µg/ml, 1 h, room temperature). First lane,
recombinant human latent TGF- (LTGF- ; 1.64 µg);
second lane, native TSP1 (nTSP; 18.6 µg).
B, proteins present in media conditioned by BAE cells were
immunoprecipitated (IP) with antibodies to LAP and analyzed
by Western blotting with a monoclonal antibody to thrombospondin.
Lane 1, conditioned media prior to immunoprecipitation;
lane 2, supernatant after immunoprecipitation; lane
3, goat polyclonal anti-LAP antibodies loaded directly onto the
gel; lane 4, media incubated with beads that were not
coupled to antibodies; lane 5, proteins immunoprecipitated
from conditioned media by incubation with anti-LAP antibodies coupled
to beads.
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LAP, Thrombospondin, and TGF-
Form Ternary Complexes That Retain
TGF-
Activity--
Since previous observations showed that
thrombospondin-1 contains associated TGF-
activity, we hypothesized
that LAP, thrombospondin, and TGF-
may form ternary complexes that
maintain bioactivity. To investigate this hypothesis, it was determined
whether removal of thrombospondin molecules that had associated LAP
resulted in depletion of TGF-
activity present in the
thrombospondin-1 preparation. Thrombospondin-associated TGF-
activity was measured prior to and following immunodepletion of
LAP-associated thrombospondin-1 with anti-LAP antibodies coupled to
Sepharose beads. Equal concentrations of protein in both the starting
and immunodepleted materials were evaluated for TGF-
activity. As
shown in Fig. 2A,
thrombospondin-1 that had been depleted of LAP by immunoprecipitation
with anti-LAP antibodies was correspondingly depleted of TGF-
activity. Immunodepletion of LAP from the thrombospondin-1 samples was
confirmed by our inability to detect LAP on Western blots of treated
samples (Fig. 2A).

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Fig. 2.
LAP is present in
thrombospondin·TGF- complexes that retain
growth factor activity. A, thrombospondin was
immunodepleted of LAP as described under "Experimental Procedures,"
and TGF- activity present in both the starting (Pre) and
final treated (Post) materials was determined in an NRK soft
agar assay. The efficiency of LAP depletion was assessed by Western
blotting (WB) of the treated thrombospondin with polyclonal
antibodies against LAP. B, thrombospondin (9 µg) was
incubated with LAP (28 ng) for 1 h at room temperature in 100 µl
of serum-free DMEM, followed by addition of active TGF- and further
incubation for an additional hour. Control proteins were incubated
under the same conditions (described under "Experimental
Procedures"). Immediately following the second incubation, all
samples were assayed for TGF- activity in the NRK assay. Results
represent means ± S.D. of triplicate determinations and are
representative of two separate experiments.
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To further investigate the hypothesis that ternary complexes containing
thrombospondin, TGF-
, and LAP retain TGF-
activity, these
proteins were incubated under conditions that allow them to form binary
and/or ternary complexes, and the resulting TGF-
activity was
measured (Fig. 2B). As expected, incubation of active TGF-
with LAP resulted in inactivation of the growth factor, indicating that the latent TGF-
complex was re-formed under these conditions. However, when TGF-
was incubated with preformed
complexes of thrombospondin and LAP (Fig. 2B), LAP failed to
inactivate TGF-
.
These observations show that LAP complexes with thrombospondin in
biological fluids and that active TGF-
, thrombospondin, and LAP can
form ternary complexes. These data also show that interactions of
thrombospondin with LAP alter the ability of this precursor portion of
TGF-
to confer latency on active TGF-
.
LAP Interacts with the KRFK Sequence in Thrombospondin-1, but Not
with the Thrombospondin-2 Sequence TRIR--
Polyclonal antibodies
raised against a peptide from the LAP amino terminus (amino acids
81-94) inhibit latent TGF-
activation by thrombospondin-1 (13).
This observation and the presence of LAP in biologically active
thrombospondin-1·TGF-
complexes suggest that the thrombospondin-1
sequence (KRFK) responsible for activation of latent TGF-
might
interact with LAP.
To test this hypothesis, we chose an approach based on
co-immunoprecipitation. When thrombospondin-1 and LAP were incubated together for 1 h at room temperature, they formed complexes that were immunoprecipitated by both polyclonal antibodies against LAP (Fig.
3, A and B,
third lanes) and monoclonal antibodies against thrombospondin (Fig. 3, C and D, third
lanes). This association between thrombospondin and LAP was
competitively inhibited by preincubation of LAP with the
thrombospondin-derived peptide KRFK. Partial inhibition occurred when
the peptide was present at a 10-100-fold molar excess relative to the
thrombospondin concentration, whereas complete inhibition was observed
when the peptide was present at a 1000-fold molar excess (Fig. 3,
A and C, fourth through seventh
lanes). These data suggest a role for the thrombospondin-1 activation sequence KRFK in LAP binding.

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Fig. 3.
Peptide KRFK inhibits binding between
thrombospondin and LAP. The interaction between stripped
thrombospondin and LAP was analyzed by immunoprecipitation
(IP) with goat polyclonal antibodies against LAP, followed
by Western blotting (WB) with anti-thrombospondin antibody
(A and B) or by immunoprecipitation with
anti-thrombospondin antibody and Western blotting with anti-LAP
antibody (C and D) as described in detail under
"Experimental Procedures." The controls include the following:
lanes 1, beads + TSP (A and B)
or beads + LAP (C and D); and lanes 2,
(beads + antibodies) + TSP (A and B) or (beads + antibodies) + LAP (C and D). In all gels, the
third lane represents TSP + LAP incubated with antibodies
and beads. A and C: fourth through
seventh lanes, LAP incubated with 0.11, 1.1, 11, or 110 µM peptide KRFK (10-104-fold molar excess
when compared with TSP), respectively, prior to incubation with TSP;
B and D: fourth through sixth
lanes, LAP incubated with 11 µM peptide KRFK, TRIR,
or KRAK, respectively, prior to incubation with TSP.
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The inability of the related sequence KRAK, which does not activate
latent TGF-
(15), to inhibit complex formation between the two
proteins (Fig. 3, B and D, sixth lane)
provides evidence that this interaction between the thrombospondin-1
sequence KRFK and LAP is specific. Furthermore, data from this
experiment also suggest that the interaction between the KRFK sequence
and LAP is relevant for the ability of thrombospondin to activate
latent TGF-
since the inactive KRFK homologue in thrombospondin-2
(TRIR) had no inhibitory effect on complex formation between
thrombospondin-1 and LAP (Fig. 3, B and D,
fifth lanes).
These data show that thrombospondin-1 binds to the LAP portion of the
latent TGF-
molecule. This binding is apparently mediated by the
TGF-
-activating sequence KRFK in thrombospondin-1 and may be part of
the mechanism by which thrombospondin-1 activates latent TGF-
.
The LSKL Sequence in LAP Interacts with the
Thrombospondin-1-derived Peptide KRFK--
To identify the sequence on
LAP responsible for binding the thrombospondin-1 sequence KRFK, we used
the molecular recognition theory as a strategy to identify sequences in
LAP complementary to the KRFK sequence in thrombospondin-1 that could
potentially form a binding site (25-28). A search based on this
theory, utilizing a computer program developed by Blalock and
co-workers (25) (parameters used described under "Experimental
Procedures"), identified only one sequence in LAP complementary to
the thrombospondin-1 sequence KRFK. This sequence (LSKL) is present in
the amino terminus of
1-LAP (positions 54-57). As a
control, this program was also used to identify sequences complementary
to the TRIR sequence in thrombospondin-2, which is homologous to the
thrombospondin-1 sequence KRFK, but does not activate latent TGF-
;
there were no sequences in LAP predicted to match the thrombospondin-2
sequence TRIR. This finding is consistent with our hypothesis that the interaction between KRFK and a specific sequence in LAP is important for the ability of thrombospondin-1 to activate latent TGF-
.
To test the hypothesis that thrombospondin-1 binds to LAP through
interactions involving the thrombospondin-1 sequence KRFK and the LAP
sequence LSKL, we used two different approaches: immunoprecipitation and affinity chromatography. Initially, we investigated the ability of
LSKL peptides to inhibit complex formation between thrombospondin-1 and
LAP. Thrombospondin·LAP complexes were isolated by
immunoprecipitation with anti-LAP or anti-thrombospondin antibodies and
detected by Western blotting with anti-thrombospondin and anti-LAP
antibodies, respectively. Fig. 4 shows
that preincubation of thrombospondin with the LAP sequence LSKL
prevented complex formation between thrombospondin-1 and LAP.
Although some inhibition could be observed when the molar concentration
of the peptide exceeded that of LAP by 10-fold (Fig. 4A),
significant inhibition was consistently seen when the peptide was used
at a 100-fold excess, with 1000-fold excess resulting in complete
inhibition of binding between LAP and thrombospondin (Fig. 4,
A and C). Furthermore, the inhibition of
LAP-thrombospondin association was specific for the LAP-derived peptide
LSKL, as indicated by the inability of the scrambled peptide SLLK to
inhibit complex formation between these two proteins, even when present
at a 1000-fold molar excess (2.8 µM) (Fig. 4, B and D, fourth lanes). Also, as shown
in Fig. 4 (B and D, fifth and
sixth lanes), inhibition occurred whether or not the LSKL sequence was accompanied by its flanking sequences (RGQILSKLRL).

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Fig. 4.
Peptide LSKL inhibits binding between
thrombospondin and LAP. The interaction between stripped
thrombospondin and LAP was analyzed by immunoprecipitation
(IP) with polyclonal goat antibodies against LAP, followed
by Western blotting (WB) with anti-thrombospondin antibody
(A and B) or by immunoprecipitation with
anti-thrombospondin and Western blotting with anti-LAP (C
and D) as described in detail under "Experimental
Procedures." The controls include the following: lanes 1,
beads + TSP (A and B) or beads + LAP
(C and D); and lanes 2, (beads + antibodies) + TSP (A and B) or (beads + antibodies) + LAP (C and D). In all gels, the
third lane represents TSP + LAP incubated with antibodies
and beads. A and C: fourth through
seventh lanes, TSP incubated with 0.28, 2.8, 28, or 280 µM peptide LSKL (10-104-fold molar excess
when compared with LAP), respectively, prior to incubation with LAP;
B and D: fourth through sixth
lanes, TSP incubated with 28 µM peptide SLLK, LSKL,
or RGQILSKLRL, respectively, prior to incubation with LAP.
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As an alternative approach, we assessed the ability of recombinant
human
1-LAP to bind to an affinity column made by
coupling the KRFK peptide to the matrix through a C-terminal cysteine
residue on the peptide. The aim of this experiment was to directly
assess whether LAP binds to the KRFK sequence in thrombospondin-1 and whether this binding could be disrupted by the LSKL peptide.
Recombinant human
1-LAP bound to the KRFK column with no
detectable LAP in the flow-through fractions, and LAP bound to the KRFK
column was specifically eluted with the LSKL peptide (Fig.
5A), suggesting that the LSKL
sequence bound to the KRFK affinity matrix. This interaction is
apparently specific since a scrambled version of the LSKL sequence,
peptide SLLK, did not elute bound LAP from the KRFK column. The failure
of LAP to bind to the TRIR affinity matrix provides further evidence
for the specificity of the LAP-KRFK interaction (Fig.
5B).

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Fig. 5.
LAP binds to a KRFK affinity column and is
specifically eluted by LSKL. LAP (10 µg/0.5 ml of PBS) was
applied to a KRFK-Sulfolink column (A) or a TRIR-Sulfolink
column (B) as described under "Experimental Procedures."
After extensive washing of unbound LAP with PBS, the columns were
sequentially treated with a 150-fold molar excess (86 µM)
of the scrambled peptide SLLK, followed by a 150-fold molar excess of
peptide LSKL. In B, elution with LSKL was followed by a wash
with a 150-fold molar excess of TRIR. Fractions (0.25 ml each fraction,
64 µl/fraction loaded onto the gel) were collected and analyzed for
the presence of LAP by Western blotting (WB) with goat
polyclonal antibodies against LAP (1 µg/ml, 1 h, room
temperature). The secondary antibody used was rabbit anti-goat IgG
(0.08 µg/ml, 1 h, room temperature). Fractions 3-5 were eluted
with peptide SLLK, LSKL, or TRIR, as indicated. Pre,
starting material; FT, flow-through fraction.
|
|
Based on these results, we conclude that the binding between
thrombospondin-1 and LAP is mediated by interactions involving the KRFK
sequence in the thrombospondin-1 molecule and the LSKL sequence present
in LAP. The role of the LSKL sequence appears to be dependent on this
specific amino acid sequence and is not simply charge-based since the
scrambled peptide SLLK is unable to prevent LAP-KRFK
interactions. Furthermore, the observation that LAP fails to
bind to a TRIR affinity matrix is consistent with the
hypothesis that interactions of the KRFK sequence with LAP are
important to the mechanism of latent TGF-
activation by
thrombospondin-1.
Activation of Recombinant or Endothelial Cell-secreted Latent
TGF-
by Thrombospondin Is Inhibited by Peptide LSKL--
To test
the hypothesis that interactions involving the KRFK sequence in the
thrombospondin type 1 repeats and the LSKL sequence in LAP are
important for activation of latent TGF-
by thrombospondin, we tested
the ability of peptide LSKL to competitively inhibit activation. In
these experiments, thrombospondin-1, peptide 246 (KRFKQDGGWSHWSPWSS),
or peptide KRFK (all at 11 nM) was preincubated with
increasing concentrations of LSKL (from 1 nM to 10 µM) prior to incubation with latent TGF-
. Activation
of latent TGF-
by either thrombospondin or KRFK-containing peptides
was inhibited by peptide LSKL in a concentration-dependent
manner (Fig. 6). LSKL alone did not
activate latent TGF-
, and it did not affect the ability of NRK cells
to respond to active TGF-
(data not shown). In experiments examining
thrombospondin-dependent activation of latent TGF-
secreted into the conditioned medium of BAE cells, peptide LSKL, but
not a control scrambled peptide (SLLK), similarly inhibited
thrombospondin-dependent activation of latent TGF-
(Fig.
7). These data support the hypothesis
that interactions involving the KRFK sequence in thrombospondin-1 and
the LSKL sequence in LAP play an essential role in activation of latent
TGF-
by thrombospondin.

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Fig. 6.
Peptide LSKL inhibits recombinant latent
TGF- activation by thrombospondin-1 and
thrombospondin-1-derived peptides. Stripped TSP1 (closed
circles), peptide 246 (open circles), or KRFK
(closed squares), all used at 11 nM, was
incubated with recombinant human latent TGF- 1 (2 nM) in the absence or presence of increasing concentrations
of peptide LSKL for 1 h at 37 °C. Samples were tested for
TGF- activity in the NRK colony formation assay. LSKL by itself
(closed triangles) is representative of the negative control
(i.e. assay performed in the presence of epidermal growth
factor and in the absence of TGF- ). Results are expressed as
means ± S.D. of triplicate determinations.
|
|

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Fig. 7.
Peptide LSKL blocks TSP-mediated activation
of latent TGF- secreted by BAE cells.
Approximately 24 h after seeding, BAE cells (1 × 105 cells/25-cm2 flask) were washed and
incubated for ~24 h in the presence of DMEM containing 2% fetal
bovine serum (2 ml/flask). Conditioned media from five flasks were
harvested, pooled together, and incubated with or without 11 nM TSP1 in the presence or absence of increasing
concentrations of peptide LSKL or control peptide SLLK (1.5 h,
37 °C). After incubation, samples were tested for TGF- activity
in an NRK colony formation assay. The solid bar represents
the number of colonies in control wells containing conditioned
medium that was not treated with TSP1. Results from controls in
which conditioned media were incubated with the peptides in the
absence of TSP1 did not differ from those obtained with
conditioned medium by itself (data not shown). Results are expressed
as means ± S.D. of triplicate determinations.
|
|
Thrombospondin-1 Activates TGF-
2 in an
LSKL-dependent Manner--
The LSKL sequence is conserved
in all TGF-
isoforms (29-34). This suggests that if
thrombospondin-1 activation of latent TGF-
is mediated via
interactions with LAP that involve the LSKL sequence, all isoforms of
TGF-
should be subject to activation by thrombospondin-1. Therefore,
the ability of thrombospondin-1 to activate recombinant latent
TGF-
2 expressed by Chinese hamster ovary cells was
tested. Purified latent TGF-
2 was incubated with
equimolar concentrations (11 nM) of stripped
thrombospondin-1, peptide 246 (KRFKQD- GGWSHWSPWSS), or KRFK and
then tested for TGF-
activity (Fig.
8). Treatment of latent
TGF-
2 with either stripped thrombospondin-1 or
thrombospondin-1 peptides resulted in a 5-fold increase in TGF-
activity as compared with untreated latent TGF-
2, which
was similar to the activation obtained by acid treatment of the latent
complex (data not shown). When latent TGF-
2 was
incubated with stripped thrombospondin-1 or thrombospondin-1 peptides
in the presence of 10 µM peptide LSKL, however,
activation was totally inhibited. These data show that thrombospondin-1
activates latent TGF-
2 and that this activation, like
that of latent TGF-
1, involves both the KRFK
sequence in thrombospondin and the LSKL sequence in LAP.

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Fig. 8.
Thrombospondin activates recombinant latent
TGF- 2. TSP1, peptide 246, or
KRFK (all at 11 nM) was incubated with recombinant latent
TGF- 2 for 1 h at 37 °C in the presence or
absence of LSKL (10 µM). Recombinant latent
TGF- 2 treated with 1 N HCl was used as a
positive control for activation (data not shown). Samples were tested
for TGF- activity by NRK colony formation assay in soft agar.
Results are expressed as means ± S.D. of triplicate
determinations.
|
|
 |
DISCUSSION |
The mechanism previously proposed for activation of latent TGF-
by thrombospondin-1 involves multiple interactions between the two
molecules (15). The WXXW sequence present in type 1 repeats
of thrombospondin-1 enhances the molar effectiveness of activation
mediated by peptides containing this sequence C-terminal to the KRFK
activation sequence. Peptides containing the WXXW sequence
can competitively inhibit binding of the active portion of TGF-
to
thrombospondin, although the WXXW sequence in itself does
not activate latent TGF-
. The function of this interaction has not
yet been clearly defined; however, it is felt that the WXXW
motifs in the type 1 repeats may act as "docking sites" to facilitate interactions of thrombospondin with the latent TGF-
complex. The second interaction involves a sequence unique to the
thrombospondin-1 isoform, K412RFK415, which is
responsible for activation of the latent TGF-
complex (15). We now
show that LAP in the latent TGF-
complex is recognized by the KRFK
sequence in thrombospondin-1. Furthermore, we have identified a
sequence (LSKL) at the amino terminus of LAP that is important for
LAP-KRFK interactions and modulation of latent TGF-
activation by
thrombospondin-1.
Formation of the latent TGF-
complex involves structural changes in
both LAP and the mature peptide (23). Based on our previous work, we
postulated that activation of latent TGF-
by thrombospondin also
involves a change in the conformation of the inactive complex (13-15).
The role of LAP in the activation process and its fate following
activation were, however, unknown. These data now show that LAP can
remain associated with the thrombospondin·TGF-
complex without
inhibiting the activity of thrombospondin-associated TGF-
. The
physiological significance of the continued association of LAP with
thrombospondin-1 following activation is not entirely clear. However,
based on our observation that LAP associated with thrombospondin is
unable to confer latency on active TGF-
(Fig. 2B), it is
reasonable that the LAP-thrombospondin association following activation
modulates the persistence of TGF-
activity by preventing refolding
of the complex and inactivation of TGF-
. It remains to be determined
whether LAP-thrombospondin-active TGF-
complexes are deposited in
the extracellular matrix or processed differently by TGF-
receptors.
We took advantage of the fact that the thrombospondin-1 gene has been
sequenced to deduce the putative site for thrombospondin-1 binding in
the LAP molecule by utilizing the molecular recognition theory (28,
36). According to this concept, translation of complementary DNA
strands predicts sequences that have exactly complementary hydropathic
profiles and that could thus function as complementary binding sites.
Examples of protein-protein interactions in which this theory has been
useful include ACTH-ACTH receptor (27), fibronectin-integrin (37, 38),
and interleukin-1
-type I receptor (39), among others. Applying this
principle to the interaction between the thrombospondin-1 sequence KRFK
and the LAP molecule, we identified the LSKL sequence in LAP of
TGF-
1-5 as the putative binding site for
thrombospondin-1. These data show that the LSKL sequence in LAP is
indeed involved in the interaction of LAP with thrombospondin-1 and
that the LSKL peptide inhibits activation of both latent
TGF-
1 and TGF-
2 by thrombospondin-1. More
direct approaches are currently being investigated to determine whether
the LSKL sequence is indeed the actual binding site for thrombospondin.
Although the overall degree of conservation among the proregions of the
various TGF-
isoforms is only 30-45%, the LSKL sequence is
conserved in all five TGF-
isoforms (29-34). Conversely, this sequence is absent in other members of the TGF-
superfamily, including Drosophila decapentaplegic protein, bone
morphogenic proteins, activins/inhibins, VGR-1, and dorsalin
(GenBankTM Data Bank search). The biological relevance of
the conserved nature of the LSKL sequence among the different LAPs is
made even more apparent by the fact that different TGF-
isoforms are
products of distinct genes, located on different chromosomes (1). It remains to be determined whether thrombospondin-1 can indeed activate latent TGF-
3-5 or whether there are additional
determinants in LAP that regulate thrombospondin's ability to activate
latent TGF-
. Nevertheless, the conservation of LSKL suggests that
this is an important sequence for regulation of activation of all
TGF-
isoforms by thrombospondin-1. This interpretation is supported by the observation that in vivo, increased thrombospondin
expression is frequently associated with increased TGF-
activity
(40-42). Since thrombospondin-1 is an early response gene that is
rapidly up-regulated in response to a number of serum factors (7, 43, 44), it is possible that regulation of thrombospondin-1 expression by
these factors also results in regulation of the activity of all
mammalian forms of TGF-
.
The interaction between KRFK and LAP does not appear to be dependent
solely on electrostatic forces. Peptide LSKL effectively prevents LAP
from binding to thrombospondin-1 (Fig. 4), disrupts the binding of LAP
to KRFK (Fig. 5), and inhibits latent TGF-
activation by
thrombospondin-1 (Figs. 6 and 7), whereas the scrambled peptide SLLK,
which retains the same overall charge, has no effect on
LAP-thrombospondin-1 or LAP-KRFK binding or the ability of thrombospondin-1 to activate latent TGF-
(Figs. 4, 5, and 7). Furthermore, activation of latent TGF-
by thrombospondin-1 is not
inhibited by the presence of 0.5 M NaCl (15). The regions surrounding the LSKL sequence in all latent TGF-
isoforms are, however, considerably charged (29-34) and may be responsible for electrostatic interactions involved in the stabilization of the latent
TGF-
complex, consistent with the hypothesis that electrostatic interactions are important for maintenance of TGF-
latency. Although other approaches will be needed to determine whether the LSKL and KRFK
sequences form the actual binding site between LAP and thrombospondin,
we believe that the data presented here are sufficient to allow us to
propose that there is minimally a sequence-specific interaction between
thrombospondin-1 and LAP, involving KRFK and LSKL. Additional
conformational factors may also be important for the interaction of
these two proteins. This is consistent with recent work of Gentry and
co-workers (45), who showed that only dimeric, but not monomeric,
1-LAP binds to immobilized thrombospondin-1. It should
be noted that there may be certain conformational restraints that
preclude complex formation, as Bailly et al. (35) reported that LAP and thrombospondin failed to bind each other when assayed using a plasmon resonance approach. We therefore suggest that sequence-
and conformation-dependent interactions between
thrombospondin-1 and LAP cause a rearrangement of LAP, which disrupts
the electrostatic interactions between LAP and the active domain, thus
converting the latent complex into a biologically active molecule.
These new findings are significant in that they further our
understanding of the mechanisms involved in activation of latent TGF-
. In doing so, they provide us with new tools (LSKL-containing peptides) to modulate in vivo TGF-
activity in situations
such as fibrosis, in which regulation of TGF-
activity would be
beneficial. Supporting evidence for this suggestion is offered by our
recent observation that thrombospondin and its derived peptides play a
significant role in the regulation of latent TGF-
activation in vivo (46). In that study, we showed that
TGF-
1 and thrombospondin-1 knockout mice have similar
histological abnormalities in nine organ systems and that treatment of
thrombospondin-1 knockout pups with KRFK peptides resulted in reversion
of the lung and pancreatic phenotypes and detection of active TGF-
in situ. Furthermore, treatment of wild-type pups with
peptide LSKL in vivo resulted in phenotypic alterations
similar to those observed in the TGF-
null animals. These
observations indicate that the interaction between the KRFK sequence in
thrombospondin and the LSKL sequence in LAP, described here as
important for the regulation of TGF-
activity in vitro,
is also important for the regulation of TGF-
activity in
vivo.
 |
ACKNOWLEDGEMENTS |
We are thankful to Drs. Jeffrey Greenwood,
Victor Darley-Usmar, and J. Edwin Blalock for helpful discussions. We
also thank Dr. Patricia Segarini and Celtrix Corp. for the generous
gift of latent TGF-
2 and Manuel A. Pallero for keeping
the lab running smoothly.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant HL50061 (to J. E.-M.-U.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Both authors contributed equally to this work.
¶
Recipient of American Heart Association Grant 96015140. To
whom correspondence should be addressed: Dept. of Pathology, Volker Hall, G038, University of Alabama at Birmingham, 1670 University Blvd.,
Birmingham, AL 35294-0019. Tel.: 205-975-9371; Fax: 205-934-1775; E-mail: ribeiro{at}path.uab.edu.
Supported by a predoctoral fellowship from the Molecular and
Cellular Graduate Program, Department of Pathology, University of
Alabama at Birmingham, and by National Institutes of Health Grant HL50061.
**
Present address: Southeast Poultry Research Lab., USDA-ARS, 934 College Station Rd., Athens, GA 30604.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
LAP, latency-associated peptide;
NRK, normal rat kidney;
BAE, bovine aortic endothelial;
DMEM, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
TSP, thrombospondin;
ACTH, adrenocorticotropic hormone.
 |
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