From the Departments of Pathology,
¶ Biomedical Engineering, and
Biochemistry,
University of Virginia Health Sciences Center,
Charlottesville, Virginia 22908
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ABSTRACT |
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2-Macroglobulin
(
2M) functions as a major carrier of transforming growth
factor-
(TGF-
) in vivo. The goal of this
investigation was to characterize the TGF-
-binding site in
2M. Human
2M, which was reduced and
denatured to generate 180-kDa subunits, bound TGF-
1, TGF-
2, and
NGF-
in ligand blotting experiments. Cytokine binding was not
detected with bovine serum albumin that had been reduced and alkylated,
and only minimal binding was detected with purified murinoglobulin. To
localize the TGF-
-binding site in
2M, five cDNA
fragments, collectively encoding amino acids 122-1302, were expressed
as glutathione S-transferase (GST) fusion proteins. In
ligand blotting experiments, TGF-
2 bound only to the fusion protein
(FP3) that includes amino acids 614-797. FP3 bound
125I-TGF-
1 and 125I-TGF-
2 in solution,
preventing the binding of these growth factors to immobilized
2M-methylamine (
2M-MA). The
IC50 values were 33 ± 5 and 26 ± 6 nM for TGF-
1 and TGF-
2, respectively; these values
were comparable with or lower than those determined with native
2M or
2M-MA. A GST fusion protein that
includes amino acids 798-1082 of
2M (FP4) and purified
GST did not inhibit the binding of TGF-
to immobilized
2M-MA. FP3 (0.2 µM) neutralized the
activity of TGF-
1 and TGF-
2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3
also increased NO synthesis by RAW 264.7 cells, mimicking an
2M activity that has been attributed to the
neutralization of endogenously synthesized TGF-
. Thus, we have
isolated a peptide corresponding to 13% of the
2M
sequence that binds TGF-
and neutralizes the activity of TGF-
in
two separate biological assays.
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INTRODUCTION |
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Human 2-macroglobulin
(
2M)1 is a
718-kDa glycoprotein that was originally characterized as a broad
spectrum proteinase inhibitor (1). The structure of
2M
consists of four identical subunits, each with 1451 amino acids (2).
The subunits are linked into dimers by disulfide bonds and into intact
homotetramers by noncovalent interactions (3, 4). Proteinases react
with
2M by cleaving any of a number of susceptible
peptide bonds in the "bait region," which includes amino acids
666-706 (1, 3, 5). Bait region cleavage causes
2M to
undergo a major conformational change, which effectively "traps"
the attacking proteinase in a complex that is nondissociable, even when
the proteinase and the inhibitor are not covalently linked (1, 6-8).
Conformational change also reveals binding sites for the
2M receptor/low density lipoprotein receptor-related
protein (LRP) (9). These binding sites have been localized to 18-kDa
peptides at the C terminus of each
2M subunit; Lys-1370
and Lys-1374 play particularly important roles (10-13).
Like the complement components, C3 and C4, each 2M
subunit contains a novel thiol ester bond, which is formed from the
side chains of Cys-949 and Glu-952 (14-16). The thiol esters may be instrumental in determining the conformational state of
2M (17, 18). When
2M reacts with a
proteinase, the thiol esters emerge from within hydrophobic,
solvent-restricted clefts and are cleaved by nucleophiles or
H2O (14, 18). Small primary amines, such as methylamine,
penetrate the hydrophobic clefts and react with
2M thiol
esters independently of proteinases, inducing an equivalent or nearly
equivalent conformational change (6, 7).
In addition to its activity as a proteinase inhibitor,
2M functions as a major carrier and regulator of certain
cytokines, including isoforms of the transforming growth factor-
(TGF-
) family. O'Connor-McCourt and Wakefield (19) first identified
2M as a physiologically significant carrier of TGF-
in human serum (19). Their studies demonstrated that nearly all of the TGF-
1 in serum is associated with
2M and that the
bound TGF-
1 is inactive. Huang et al. (20) confirmed the
role of
2M as a TGF-
-carrier and demonstrated that
the TGF-
binding activity of
2M depends on its
conformational state.
More recent studies have demonstrated the function of 2M
as a TGF-
-carrier in animal model systems. When radioiodinated TGF-
1 is injected intravascularly in mice, the cytokine is cleared rapidly at first; however, this is followed by a slow clearance phase,
during which time the TGF-
is almost entirely
2M-associated (21-23). In cell culture systems,
2M neutralizes both exogenously added and endogenously
synthesized TGF-
(24-28). Neutralization of endogenously
synthesized TGF-
results in altered gene expression, including
greatly increased expression of inducible nitric-oxide synthase by
murine macrophages and increased expression of platelet-derived growth
factor
-receptor by vascular smooth muscle cells (27, 28).
2M gene knockout mice demonstrate increased tolerance to
endotoxin challenge (29); this characteristic is most likely explained
by the enhanced function of TGF-
as an immunosuppressant, in the
absence of
2M (30). The function of
2M as
a significant modulator of TGF-
activity in vivo and
in vitro has prompted us to elucidate the
2M-TGF-
interaction on a molecular level.
Binding of TGF- to
2M is initially noncovalent and
reversible; however, the complex can become covalently stabilized as a
result of thiol-disulfide exchange (23). The latter reaction is
observed primarily with conformationally altered
2M,
since native
2M lacks free thiol groups (23, 31, 32). We
have used a number of complementary methods to determine equilibrium dissociation constants (KD) for the interaction of
TGF-
with
2M (23, 31, 33). The KD
values for the binding of TGF-
1 and TGF-
2 to native
2M are 300 and 10 nM, respectively; the
KD values for the binding of TGF-
1 and TGF-
2 to methylamine-modified
2M (
2M-MA) are 80 and 10 nM, respectively. These binding constants accurately
predict the ability of
2M to neutralize TGF-
in cell
culture systems (26, 30, 34, 35).
The mechanism by which 2M binds cytokines remains
unclear. Early studies, suggesting a prominent role for the thiol
ester-derived Cys-residues, were not confirmed for TGF-
1 and
TGF-
2 (32). When
2M-MA was treated with papain to
release the 18-kDa receptor binding domains, the TGF-
-binding
activity remained with the residual 600-kDa
2M fragment
(36). Thus, the cytokine- and LRP-binding sites are not co-localized.
The goal of this study was to determine whether TGF-
-binding can be
localized to a specific region in the structure of
2M.
An alternative model is that the central cavity in the structure of
2M, which serves as the proteinase trap, also
nonspecifically binds cytokines. Arguments in support of the
alternative model include the complex quaternary structure of
2M, the known trapping mechanism by which
2M interacts with proteinases, and the large number of
structurally unrelated cytokines that have been reported to associate
with
2M (37).
In this study, we present evidence demonstrating, for the first time,
that TGF- binding to
2M is not dependent on
2M quaternary structure and thus not dependent on the
2M trap. Furthermore, we localize the TGF-
-binding
site to a single 20-kDa peptide that also contains the
2M bait region. The 20-kDa peptide, when expressed as a
GST fusion protein, binds both TGF-
isoforms with equivalent or
increased affinity, compared with intact
2M. The peptide
also neutralizes the activity of TGF-
in endothelial cell
proliferation assays and macrophage NO synthesis experiments. Thus,
this 20-kDa peptide mimics the TGF-
-regulatory activity of intact
homotetrameric
2M.
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MATERIALS AND METHODS |
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Reagents and Proteins--
TGF-2 was purchased from Genzyme
(Cambridge, MA). TGF-
1 was from R & D Systems (Minneapolis, MN).
Nerve growth factor-
(NGF-
) was purified from male mouse
submaxillary glands by the method of Darling and Shooter (38).
Methylamine HCl, chloramine T, iodoacetamide (IAM), dithiothreitol
(DTT), isopropylthio-
-D-galactoside, N-octyl
glucopyranoside, glutathione S-transferase (GST),
glutathione, anti-GST IgG fraction of antiserum, and bovine serum
albumin (BSA) were from Sigma. Na125I was from Amersham
Pharmacia Biotech. pGEX-3X, pGEX-2T, and prepacked glutathione-Sepharose-4B columns were from Amersham Pharmacia Biotech.
Immulon 2 microtiter plates were from Dynatech Laboratories (Chantilly,
VA). Polyvinylidene fluoride (PVDF) and nitrocellulose membranes were
from Millipore Corp. IODO-GEN was from Pierce. RPMI 1640, Dulbecco's
modified Eagle's medium (DMEM), and Trypsin-EDTA were from Life
Technologies, Inc. Fetal bovine serum (FBS) was from Hyclone
Laboratories. Acidic fibroblast growth factor and basic fibroblast
growth factor were from Promega.
-Macroglobulins and Related Derivatives--
Human
2M was purified from plasma by the method of Imber and
Pizzo (39). Murinoglobulin (MUG) was purified from the plasma of CD-1
female mice as described previously (30). SDS-PAGE analysis of purified
MUG revealed a single band with an apparent mass of 180 kDa.
2M-MA was prepared by dialyzing human
2M
against 200 mM methylamine-HCl in 50 mM
Tris-HCl, pH 8.2, for 12 h at 22 °C followed by extensive
dialysis against 20 mM sodium phosphate, 150 mM
NaCl, pH 7.4 (PBS), at 4 °C. Complete modification of native
2M by methylamine was confirmed by loss of trypsin
binding activity (greater than 96%) (40) and by the characteristic
increase in electrophoretic mobility, when analyzed by nondenaturing
PAGE (41, 42). Monomeric
2M was prepared by exposing the
native form of the protein to a high concentration of DTT (2 mM) under nondenaturing conditions, as described by Moncino
et al. (43). Incompletely dissociated
2M was
separated from the monomers by FPLC on Superose-6. Monomeric
2M, which is prepared as described, does not reassociate
at 22 °C (44).
Preparation of Constructs Encoding GST-2M-Peptide
Fusion Proteins--
The human
2M cDNA in
pAT153/PvuII/8 (pAT-
2M) was obtained from the
ATCC (16). Restriction digest analysis revealed an additional
SacI cleavage site, which was not predicted by the published
sequence (16), due to a single base substitution at nucleotide 2431 (C
T). To generate a construct encoding GST-
2M peptide
fusion protein-1 (FP1), a fragment from pAT-
2M that
encodes amino acids 122-415 was excised with BstXI,
blunt-ended with T4 DNA polymerase, and ligated into pGEX-3X at the
SmaI site. The construct encoding FP2 was prepared by
digesting pAT-
2M with EcoRI and
NsiI, to yield a partial cDNA encoding amino acids
364-712, which was further digested with SacI, to generate
a cDNA encoding amino acids 364-613. This fragment was blunt-ended
and ligated into pGEX-2T at the SmaI site. Constructs
encoding FP3 and FP4 were prepared by isolating cDNAs, from a
SacI digest of pAT-
2M, corresponding to amino
acids 614-797 and 798-1082, respectively. These cDNAs were
blunt-ended and ligated into the SmaI site of pGEX-2T. The
construct encoding FP5 was prepared by digesting pAT-
2M
with XhoI and PstI. A resulting cDNA, which
encodes amino acids 1053-1302, was blunt-ended and ligated into
pGEX-2T at the SmaI site. Restriction digest analysis of the
five constructs confirmed that the
2M cDNA inserts
were in the correct orientation. Fig. 1
shows the relationship of the five peptides to the intact structure of
2M.
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Purification of GST-2M Peptide Fusion
Proteins--
BL21 cells harboring pGEX-
2M-peptide
expression constructs were induced with 0.1 mM
isopropylthio-
-D-galactoside for 3 h at 37 °C,
harvested by centrifugation, and resuspended in 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 1 mM EGTA, pH 8.0. Nearly pure fusion protein preparations
were generated by treating bacterial suspensions with 1 mg/ml lysozyme
for 15 min on ice. The suspensions were then sonicated and subjected to
centrifugation at 12,000 × g for 10 min. All five
fusion proteins remained in the insoluble fraction. These fractions
were suspended in 10 mM deoxycholate for 2 h,
sonicated, and subjected to a second centrifugation step. The fusion
proteins, which again remained in the insoluble fractions, were
solubilized by sonication in 2.0% SDS. To block free sulfhydryls, each
fusion protein was reacted with 1 mM IAM in SDS for 2 h at 25 °C. The IAM was then removed by dialysis. Final fusion
protein preparations were stored in SDS. Protein concentrations were
determined by the bicinchoninic acid (BCA) method.
Ligand Blotting--
Native 2M,
2M-MA, MUG, and BSA were incubated in 2% SDS, in the
presence or absence of 1 mM DTT, for 30 min at 37 °C. To block free sulfhydryls, some samples were treated with 5 mM
IAM for 2 h at 25 °C. Equivalent amounts of each protein (5 µg) were subjected to SDS-PAGE on 5% slabs. IAM-treated
GST-
2M-peptide fusion proteins were subjected to
SDS-PAGE as well. All samples were electrotransferred to PVDF
membranes. The membranes were blocked with 5% milk and 0.1% Tween 20 in PBS for 12 h at 4 °C and then rinsed with 0.1% Tween 20 in
PBS (PBS-T). Membranes with native
2M,
2M-MA, MUG, and BSA were probed with
125I-TGF-
2 (20 pM),
125I-TGF-
1 (20 pM), or
125I-NGF-
(50 pM) for 2 h at 25 °C;
membranes with the five GST-
2M peptide fusion proteins
were probed with 125I-TGF-
2. The TGF-
1 and TGF-
2
were radioiodinated, to a specific activity of 100-200 µCi/µg, as
described previously (46). NGF-
was radioiodinated with IODO-GEN, to
a specific activity of 2-5 µCi/µg, using the method recommended by
the manufacturer. To determine whether TGF-
binding to FP3 is
noncovalent and specific, membranes containing immobilized FP3 (0.5 µg) were incubated with 125I-TGF-
1 (0.25 nM) or 125 I-TGF-
2 (0.25 nM) in
the presence of unlabeled TGF-
1 (200 nM), unlabeled
TGF-
2 (200 nM), or solution phase FP3 (1.0 µM). After washing the membranes with PBS-T, bound
radioligands were detected by PhosphorImager analysis (Molecular
Dynamics, Inc., Sunnyvale, CA).
Western Blot Analysis--
GST-2M peptide fusion
proteins were subjected to SDS-PAGE and electrotransferred to
nitrocellulose membranes. The membranes were blocked with 5% milk in
PBS-T for 12 h at 4 °C, incubated with a polyclonal antibody
that recognizes GST and then with peroxidase-conjugated goat
anti-rabbit IgG. Binding of secondary antibody was detected by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Binding of 125I-TGF-2 to FP3 and FP4 as Determined
by FPLC--
125I-TGF-
2 (0.5 nM) was
incubated with FP3 or FP4 (0.5 µM) in PBS for 30 min at
37 °C. The FP3 and FP4 were purified by glutathione affinity
chromatography, treated with IAM, and free of detergents. 125I-TGF-
2-fusion protein complexes were separated from
free 125I-TGF-
2 by FPLC on prepacked Superose-12
columns. The flow rate was 0.4 ml/min. Elution of FP3 or FP4 was
detected by monitoring the absorbance at 280 nm.
125I-TGF-
2 was detected in elution fractions using a
-counter. To calibrate the FPLC, the following proteins were
subjected to chromatography on the same column: soybean trypsin
inhibitor (Mr ~21,500,
Ve of 14.1 ml), ovalbumin
(Mr ~45,000, Ve of 12.9 ml), BSA (Mr ~66,000,
Ve of 12.1 ml), and BSA dimer
(Mr ~132,000, Ve of
10.9 ml).
125I-TGF- Binding to Immobilized
2M-MA--
2M-MA (1 µg in 100 µl)
was incubated in 96-well microtiter plates for 4 h at 22 °C, as
described previously (33). This procedure results in the immobilization
of approximately 90 fmol of
2M-MA. The wells were washed
three times with PBS-T and blocked with PBS-T for 16 h at 4 °C.
As a control, some wells were blocked with PBT-T without first
immobilizing
2M-MA. 125I-TGF-
1 or
125I-TGF-
2 (0.1 nM) was incubated with the
immobilized
2M-MA in the presence of increasing
concentrations of FP3 or FP4 (4-250 nM) for 1 h at
22 °C. The fusion proteins were purified and detergent-free. The
wells were then washed three times with PBS-T.
125I-TGF-
, which was associated with the immobilized
phase, was recovered in 0.1 M NaOH, 2% SDS and quantitated
in a
-counter. Results were analyzed by plotting the specific
binding of 125I-TGF-
versus the log of the
fusion protein concentration. In these experiments, the concentration
of TGF-
(TGF-
1 or TGF-
2) was at least 100-fold lower than the
KD for TGF-
-binding to immobilized
2M-MA. Thus, TGF-
-binding was linearly related to the
free TGF-
concentration ([
F]), according to the
following equation: B = (Bmax/KD)[
F].
In the presence of a fusion protein (FP) that binds TGF-
, the total
concentration of TGF-
([
T]) was related to
[
F], at equilibrium, as follows: [
T] = [
F](1 + [FP]/KI). If the fusion
protein-TGF-
complex did not bind to immobilized
2M-MA, then TGF-
-binding was reduced by 50% (the
IC50) when [
T]/[
F] = 2, and the fusion protein concentration that yielded the IC50
was equal to the KI.
Endothelial Cell Proliferation Assays--
FBHE cells were
cultured in DMEM supplemented with 10% FBS, 20 ng/ml acidic fibroblast
growth factor, and 80 ng/ml basic fibroblast growth factor and passaged
at subconfluence with trypsin-EDTA. To perform proliferation assays,
the cells were plated at a density of 2 × 104/well
(24-well plates) in DMEM supplemented with 0.2% FBS. The cells were
pulse-exposed to TGF-1 or TGF-
2 (10 pM), in the
presence and absence of FP3 or FP4 (200 nM), for 1 h.
The fusion proteins were preincubated with the TGF-
for 15 min and
then added to the cultures. At the completion of an incubation, the
cultures were washed three times with serum-free DMEM and then allowed to incubate in DMEM with 0.2% FBS for 30 h.
[3H]Thymidine was added for an additional 18 h; the
cells were then harvested, and [3H]thymidine
incorporation was quantitated.
Nitric Oxide Synthesis--
NO synthesis by RAW 264.7 cells was
quantitated by measuring the stable NO oxidation product, nitrite, in
conditioned medium, as described previously (47). Cells were plated at
a density of 104/well in 96-well plates and cultured in
RPMI 1640 with 10% FBS for 24 h and then in RPMI 1640 without
serum (SFM) for an additional 24 h. 2M-MA, FP3,
FP4, or GST was added separately to the cultures in SFM. The fusion
proteins were purified and detergent-free. After 24 h, conditioned
medium (100 µl) was recovered, and nitrite was measured. We
previously demonstrated that
2M increases RAW 264.7 cell
NO synthesis by neutralizing endogenously produced TGF-
(27). The
2M-induced increase in RAW 264.7 cell NO synthesis is
inhibited by the nitric-oxide synthase inhibitor,
NG-monomethyl-L-arginine.
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RESULTS |
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Ligand Blot Analysis of 125I-TGF- Binding to
2M--
Native
2M,
2M-MA,
and BSA were denatured in SDS (with or without reductant), subjected to
SDS-PAGE, and electrotransferred to PVDF membranes. Some samples were
treated with IAM prior to electrophoresis. The membranes were stained
with Coomassie Blue, demonstrating nearly equivalent electrotransfer of
the three proteins (results not shown). Unreduced
2M
migrated as a single band with an apparent mass of 360 kDa, as
expected; reduced
2M migrated as a single major band
with an apparent mass of 180 kDa (3). Methylamine treatment did not
alter the mobility of
2M (14, 15, 48).
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Ligand Blot Analysis of the Binding of 125I-TGF-2 to
MUG--
MUG is a monomeric murine homologue of human
2M. Although tetrameric murine
2M, in its
native form, and human
2M bind TGF-
1 and TGF-
2
similarly, MUG does not bind either TGF-
isoform with significant
affinity (KD ~1.0 µM) (30). Thus, we
compared the binding of 125I-TGF-
2 to human
2M and MUG, as another test of the validity of the
ligand blotting method. As shown in Fig.
3, only trace levels of
125I-TGF-
2 bound to MUG, and the amount of binding was
decreased when the MUG was treated with IAM. These results support the
hypothesis that ligand blotting is a valid method for the analysis of
cytokine binding to
-macroglobulins. Apparently, MUG does not
contain a cryptic TGF-
-binding site that is exposed by SDS
treatment.
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TGF-2-Binding to GST-
2M Peptide Fusion
Proteins--
The five fusion proteins were subjected to SDS-PAGE and
electrotransferred to PVDF. The electrophoretic mobility of the major Coomassie-stained band, in each preparation, indicated a molecular mass
that was identical to the mass of the monomeric fusion protein predicted by the cDNA sequence (Fig.
4). Western blot analysis with a
GST-specific antibody confirmed that the major band in each lane was a
GST fusion protein. The low mobility bands also bound GST-specific
antibody and thus most likely represent SDS-insensitive fusion protein
aggregates. In ligand blotting experiments, only FP3 bound
125I-TGF-
2. Since all five fusion proteins were
IAM-treated, free sulfhydryl groups in FP3 did not account for the
125I-TGF-
2 binding. FP1, FP2, FP4, FP5, and purified GST
(not shown) did not bind 125I-TGF-
2.
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Binding of 125I-TGF-2 to FP3 in
Solution--
125I-TGF-
2 (0.5 nM) was
incubated with FP3 or FP4 (0.5 µM) in solution, in the
absence of detergents. Free and fusion protein-associated 125I-TGF-
2 were separated by FPLC on Superose-12. We
previously demonstrated that free TGF-
interacts substantially with
Superose and thus is recovered slowly at volumes that exceed the
totally included volume (36). As shown by the absorbance tracings (280 nm) in Fig. 5, FP3 and FP4 eluted at
volumes suggesting that these fusion proteins are dimers. The
Ve values were 11.4 and 11.2 ml for FP3 and FP4,
respectively, corresponding to apparent masses of 95- and 107-kDa.
Other GST fusion proteins are also expressed as noncovalent dimers
(49). Substantial amounts of radioactivity co-eluted with FP3; 42% of
the 125I-TGF-
2 was recovered with this fusion protein
(n = 2). By contrast, only 6% of the TGF-
2
co-eluted with FP4. FPLC is a nonequilibrium method for assessing
protein-protein interactions. The amount of complex detected may be
significantly lower than that which was initially present (23).
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125I-TGF--Binding to Immobilized
2M-MA--
125I-TGF-
1 and
125I-TGF-
2 (0.1 nM) were incubated in
separate
2M-MA-coated microtiter wells for 1 h;
3.4 ± 0.3 fmol of 125I-TGF-
1 and 2.6 ± 0.2 fmol of 125I-TGF-
2 bound to the immobilized
2M-MA. Unlabeled TGF-
(0.2 µM)
decreased the binding of 125I-TGF-
by greater than 75%.
125I-TGF-
recovery in the immobilized phase was
decreased by greater than 95% when the wells were not precoated with
2M-MA.
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FBHE Cell Proliferation Assays--
To determine whether
FP3-binding inhibits TGF- activity, FBHE cell proliferation assays
were performed. The cells were pulse-exposed to TGF-
1 or TGF-
2
(10 pM) for 1 h, in the presence and absence of FP3 or
FP4. [3H]Thymidine incorporation was measured 30 h
later. As shown in Table II,
[3H]thymidine incorporation was decreased 69 and 57% by
TGF-
1 and TGF-
2, respectively. No change in TGF-
activity was
observed when FP4 was included in the medium. By contrast, FP3 nearly
completely inhibited the activities of both TGF-
1 and TGF-
2,
increasing [3H]thymidine incorporation to within 3 and
6% of the control values.
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Regulation of NO Synthesis by FP3 and FP4--
2M
neutralizes TGF-
, which is synthesized and activated endogenously by
RAW 264.7 cells, and thereby induces expression of inducible
nitric-oxide synthase (27, 30). In order to determine whether FP3
neutralizes the activity of endogenously synthesized TGF-
, we
assessed the ability of the fusion protein to induce the production of
nitrite in RAW 264.7 cell-conditioned medium. As shown in Fig.
7, FP3 increased NO synthesis in a
concentration-dependent manner and, at low concentrations,
was more active than
2M-MA. We previously demonstrated
that the increase in NO synthesis, which is induced by 280 nM
2M-MA, is comparable with that observed with 10 ng/ml interferon-
(27). FP4 and purified GST (250 nM) did not increase nitrite production by the RAW 264.7 cells.
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DISCUSSION |
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The TGF- family of cytokines regulates diverse processes
including cellular growth, differentiation, wound healing, and
inflammation (for review, see Refs. 50 and 51). At the cellular level, TGF-
response is mediated by or regulated by a variety of receptors and binding proteins, including the type I and type II receptors, which
are serine/threonine kinases,
-glycan, and endoglin. TGF-
activity is also regulated by processes that alter delivery of the
active cytokine to the cell surface. For example, TGF-
is secreted
as a large latent complex that includes latency-associated peptide and
a second gene product, latent TGF-
-binding protein (52-54).
Conversion of latent TGF-
into active 25-kDa homodimer requires
dissociation of latency-associated peptide and latent TGF-
-binding
protein in reactions that may be mediated by proteinases (55),
thrombospondin (56), the mannose 6-phosphate/insulin-like growth
factor-II receptor (57) and acidic microenvironments (58). Once
activated, the 25-kDa form of TGF-
may bind to
2M, once again forming a complex that is unavailable for receptor binding.
The fate of 2M-associated TGF-
depends on the
2M conformation. Native
2M, which is the
predominant form of
2M present in the plasma and
probably in most extravascular microenvironments, binds TGF-
reversibly and noncovalently (23, 31, 32). Thus, native
2M may buffer tissues against rapid changes in TGF-
levels by binding or slowly releasing the cytokine in response to the free TGF-
concentration. Based on the KD value,
we predict that approximately 95% of the TGF-
1 in plasma is
2M-associated under equilibrium conditions, even though
TGF-
1 binds to native
2M with lower affinity than
TGF-
2 (31). Conversion of
2M into the transformed
conformation, which probably occurs most frequently at sites of
inflammation due to the increase in cellular proteinase secretion,
alters the mechanisms by which TGF-
is regulated. First, transformed
2M has free Cys residues and thus undergoes
thiol-disulfide exchange with TGF-
(23, 31, 32), eliminating the
potential for release of active cytokine. Second,
2M-proteinase complexes bind to the endocytic receptor,
LRP; bound TGF-
is internalized with the
2M-proteinase complex and probably delivered to
lysosomes (22, 36).
The goal of the present investigation was to identify the binding site
for TGF- in
2M. Our original ligand blotting
experiments, with human
2M, demonstrated that intact
quaternary structure is not necessary for TGF-
binding. Treatment of
2M with IAM did not inhibit TGF-
-binding, indicating
that free Cys residues, which arose either as a result of thiol ester
aminolysis or DTT treatment, are not involved. We also studied the
binding of TGF-
to purified MUG by ligand blotting, since
nondenatured MUG, unlike human
2M and tetrameric murine
2M, does not bind TGF-
with significant affinity
(30). MUG bound only trace levels of TGF-
in ligand blotting
studies, supporting our hypothesis that ligand blotting provides a
valid model of TGF-
-
-macroglobulin interactions that occur under
nondenaturing conditions. The inability of TGF-
and NGF-
to bind
to reduced and alkylated BSA further supports the use of ligand
blotting as a valid model system.
When the majority of the 2M cDNA was expressed in a
series of five GST fusion proteins, TGF-
-binding was localized
exclusively to FP3. The other four fusion proteins and purified GST did
not bind TGF-
. Selective binding of TGF-
to affinity-purified
FP3, and not to FP4, was demonstrated by FPLC and by
radioligand-binding competition assay. FP3 was more effective than
native
2M or
2M-MA at inhibiting TGF-
1
binding to immobilized
2M-MA. This result is intriguing
for at least three reasons. First, in comparing FP3 and intact
2M, we used the concentrations of intact
2M tetramer and FP3 monomer, although our FPLC results
suggested that FP3 is a noncovalent dimer. If, instead, we had based
the IC50 values on the concentration of the
2M "subunit," then the difference between FP3 and
intact
2M would have been 4-fold greater. Second, the
experimentally determined IC50 values accurately estimate the KI only if one molecule of competitor is
sufficient to completely prevent TGF-
-binding to immobilized
2M-MA; otherwise, the KI is lower
than the IC50. Although it is possible that two copies of
FP3 or
2M are required to neutralize TGF-
, given the
homodimeric structure of TGF-
, this possibility is considered less
likely with intact
2M, due to its large size and complex
structure, as discussed below. Also, as discussed below, our studies
suggest that tetrameric
2M may bind more than one
molecule of TGF-
. Finally, we cannot be certain that FP3 adopts a
secondary and tertiary structure that is optimal for TGF-
binding.
Taken together, these results suggest that a specific sequence in FP3
binds TGF-
with relatively high affinity. The equivalent sequence
may be partially masked within intact
2M, accounting for
the observed decrease in TGF-
binding affinity. The masking of the
TGF-
-binding site in intact
2M may also explain why
2M conformational change markedly alters TGF-
binding
affinity (31).
Human 2M and bovine
2M bind TGF-
2 with
increased affinity compared with TGF-
1 (24, 31), explaining why
TGF-
1 is preferentially active in certain cell culture assays that
require serum-supplemented medium (24-26). Danielpour and Sporn (24)
provided evidence that the
-macroglobulins from rabbit also
preferentially bind TGF-
2. By contrast, murine
2M
binds TGF-
1 and TGF-
2 with equivalent affinity (30). In this
study, we demonstrated that TGF-
1 and TGF-
2 bind to FP3 with
equivalent affinity as well. This result suggests that the isoform
specificity in TGF-
binding to certain
-macroglobulins may be due
to the ability of TGF-
2 to preferentially access the FP3-binding
site in the intact
-macroglobulin. When the structural constraints
of intact
2M are eliminated, as in FP3, isoform
specificity in TGF-
binding is no longer observed. We do not
understand why the binding site for TGF-
2 may be "less masked"
in the structure of intact human
2M compared with the binding site for TGF-
1; however, NMR and x-ray crystallography studies have demonstrated the presence of small differences in the
overall shape and structure of TGF-
1 and TGF-
2 (59-61).
In addition to the TGF--binding site, FP3 also contains the
2M bait region. Models have been developed regarding the
location of the bait region within the complex three-dimensional
structure of
2M based on electron microscopy (62, 63);
the x-ray crystal structure, which has been solved at 10-Å resolution
(64); NMR and EPR spectroscopy studies (65, 66); and fluorescence
resonance energy transfer studies (67). The overall structure of
2M resembles a hollow cylinder with a two-compartment
central cavity. In
2M-proteinase complexes, the
proteinases occupy the central cavities. The bait regions are located
within the central cavities, toward the center of the
2M
structure, and within 11-17 Å of the Cys residues (Cys-949) that form
the thiol ester bonds (64). If, in fact, the bait region and the
TGF-
-binding site are equivalent or overlapping, then the
TGF-
-binding site may be accessible only from within the
2M central cavity. TGF-
-specific antibodies fail to
recognize
2M-associated TGF-
(19, 24), supporting the
hypothesis that TGF-
occupies the central cavity; however, it is not
clear whether the
2M, which was studied in the antibody
experiments, was in the native or conformationally altered form. Thus,
the location of the FP3-binding site for TGF-
, within intact
2M, remains unresolved. The bait region is known as an
area of extreme sequence variability among
-macroglobulins from
different species (5). Since TGF-
-binding is conserved among many
-macroglobulins, with the exception of MUG (30, 31, 34), one can
argue that the bait region and TGF-
-binding site are unlikely to be
equivalent. Further studies will be necessary to determine the
relationship between these two important functional regions.
The stoichiometry of cytokine binding to 2M has been
estimated at 1:1 or 2:1 (19, 68). Our results suggest that the binding site contained within a single
2M subunit may be
sufficient to bind TGF-
. Thus, an estimate of four cytokine-binding
sites per
2M does not seem unreasonable. Limitations in
the number of cytokine-binding sites in intact
2M may
result from steric hindrance. If
2M-associated cytokines
occupy the central cavity, then the number of cytokines that bind may
be limited by the available cavity space. Of equal importance is the
possibility that a high affinity complex between
2M and
TGF-
requires that the cytokine engage two equivalent copies of FP3
on different subunits. KD values, determined by the
2M-MA immobilization method and by our previously
described BS3-cross-linking method (31, 33), assume a
single cytokine-binding site per
2M tetramer. If there
are two independent binding sites, then the KD for
each site would be increased by a factor of 2; however, our reported
binding constants may still be most useful for predicting the
cytokine-neutralizing activity of
2M in biological
assays.
In FBHE cell proliferation assays, we demonstrated that FP3 not only
binds TGF-1 and TGF-
2 but also neutralizes the activities of
these cytokines. When added to RAW 264.7 cell cultures, FP3 promoted
the accumulation of nitrite more effectively than
2M-MA. Since we previously demonstrated that the induction of NO synthesis by
2M is due to the neutralization of TGF-
(27), we
hypothesized that the increased potency of FP3 may be due to its
increased binding affinity for TGF-
1. To test this hypothesis, we
measured the secretion of TGF-
1 and TGF-
2 by RAW 264.7 cells
using isoform-specific enzyme-linked immunosorbent assays. In medium
that was conditioned for 24 h, the concentrations of active and
total (active plus latent) TGF-
1 were 2 and 10 pM,
respectively. The concentrations of active and total TGF-
2 were 1 and 4 pM, respectively. The active TGF-
levels reported
here are only slightly lower than those determined previously using an
endothelial cell growth assay (27). More importantly, the enzyme-linked
immunosorbent assays confirm that RAW 264.7 cells express both TGF-
isoforms but higher levels of TGF-
1, supporting the hypothesis that
the increased potency of FP3 reflects its increased capacity to
neutralize TGF-
1.
In summary, we have identified a single peptide from the structure of
2M that contains the binding site for TGF-
1 and
TGF-
2. The high affinity of FP3 for both TGF-
isoforms and the
substantial potency of FP3 in two TGF-
neutralization assays
suggests that the TGF-
-binding sequence may be partially masked in
intact
2M. Like TGF-
1 and TGF-
2, NGF-
bound to
dissociated
2M subunits, suggesting that intact
quaternary structure and the resulting
2M central cavity
or trap is not necessary. However, at this time, we have not determined
whether the NGF-
-binding site or the binding site for any other
cytokine is contained within FP3. The fusion proteins generated in this
study will represent excellent templates for defining other
cytokine-binding sites in
2M and for further refinement
of the TGF-
binding sequence.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA-53462.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.
§ A fellow of the American Heart Association, Virginia Affiliate.
** To whom correspondence should be addressed: University of Virginia Health Sciences Center, Depts. of Pathology and Biochemistry, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-982-0283; E-mail: SLG2T{at}VIRGINIA.EDU.
1
The abbreviations used are: 2M,
2-macroglobulin;
2M-MA,
2M-methylamine; MUG, murinoglobulin; TGF-
,
transforming growth factor-
; NGF-
, nerve growth factor-
; NO,
nitric oxide; LRP, low density lipoprotein receptor-related protein;
FBS, fetal bovine serum; IAM, iodoacetamide; DTT, dithiothreitol; BSA,
bovine serum albumin; PVDF, polyvinylidene fluoride; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; SFM,
serum-free medium; FP1-FP5, GST fusion proteins 1-5; DMEM,
Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel
electrophoresis; FPLC, fast protein liquid chromatography; FBHE cell,
fetal bovine heart endothelial cell.
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REFERENCES |
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