Localization of the Binding Site for Transforming Growth Factor-beta in Human alpha 2-Macroglobulin to a 20-kDa Peptide That Also Contains the Bait Region*

Donna J. WebbDagger §, Janice WenDagger , Larry R. Karns, Michael G. KurillaDagger , and Steven L. GoniasDagger parallel **

From the Departments of Dagger  Pathology,  Biomedical Engineering, and parallel  Biochemistry, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha 2-Macroglobulin (alpha 2M) functions as a major carrier of transforming growth factor-beta (TGF-beta ) in vivo. The goal of this investigation was to characterize the TGF-beta -binding site in alpha 2M. Human alpha 2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta 1, TGF-beta 2, and NGF-beta 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-beta -binding site in alpha 2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta 2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta 1 and 125I-TGF-beta 2 in solution, preventing the binding of these growth factors to immobilized alpha 2M-methylamine (alpha 2M-MA). The IC50 values were 33 ± 5 and 26 ± 6 nM for TGF-beta 1 and TGF-beta 2, respectively; these values were comparable with or lower than those determined with native alpha 2M or alpha 2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha 2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha 2M-MA. FP3 (0.2 µM) neutralized the activity of TGF-beta 1 and TGF-beta 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 alpha 2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta . Thus, we have isolated a peptide corresponding to 13% of the alpha 2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human alpha 2-macroglobulin (alpha 2M)1 is a 718-kDa glycoprotein that was originally characterized as a broad spectrum proteinase inhibitor (1). The structure of alpha 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 alpha 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 alpha 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 alpha 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 alpha 2M subunit; Lys-1370 and Lys-1374 play particularly important roles (10-13).

Like the complement components, C3 and C4, each alpha 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 alpha 2M (17, 18). When alpha 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 alpha 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, alpha 2M functions as a major carrier and regulator of certain cytokines, including isoforms of the transforming growth factor-beta (TGF-beta ) family. O'Connor-McCourt and Wakefield (19) first identified alpha 2M as a physiologically significant carrier of TGF-beta in human serum (19). Their studies demonstrated that nearly all of the TGF-beta 1 in serum is associated with alpha 2M and that the bound TGF-beta 1 is inactive. Huang et al. (20) confirmed the role of alpha 2M as a TGF-beta -carrier and demonstrated that the TGF-beta binding activity of alpha 2M depends on its conformational state.

More recent studies have demonstrated the function of alpha 2M as a TGF-beta -carrier in animal model systems. When radioiodinated TGF-beta 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-beta is almost entirely alpha 2M-associated (21-23). In cell culture systems, alpha 2M neutralizes both exogenously added and endogenously synthesized TGF-beta (24-28). Neutralization of endogenously synthesized TGF-beta 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 alpha -receptor by vascular smooth muscle cells (27, 28). alpha 2M gene knockout mice demonstrate increased tolerance to endotoxin challenge (29); this characteristic is most likely explained by the enhanced function of TGF-beta as an immunosuppressant, in the absence of alpha 2M (30). The function of alpha 2M as a significant modulator of TGF-beta activity in vivo and in vitro has prompted us to elucidate the alpha 2M-TGF-beta interaction on a molecular level.

Binding of TGF-beta to alpha 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 alpha 2M, since native alpha 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-beta with alpha 2M (23, 31, 33). The KD values for the binding of TGF-beta 1 and TGF-beta 2 to native alpha 2M are 300 and 10 nM, respectively; the KD values for the binding of TGF-beta 1 and TGF-beta 2 to methylamine-modified alpha 2M (alpha 2M-MA) are 80 and 10 nM, respectively. These binding constants accurately predict the ability of alpha 2M to neutralize TGF-beta in cell culture systems (26, 30, 34, 35).

The mechanism by which alpha 2M binds cytokines remains unclear. Early studies, suggesting a prominent role for the thiol ester-derived Cys-residues, were not confirmed for TGF-beta 1 and TGF-beta 2 (32). When alpha 2M-MA was treated with papain to release the 18-kDa receptor binding domains, the TGF-beta -binding activity remained with the residual 600-kDa alpha 2M fragment (36). Thus, the cytokine- and LRP-binding sites are not co-localized. The goal of this study was to determine whether TGF-beta -binding can be localized to a specific region in the structure of alpha 2M. An alternative model is that the central cavity in the structure of alpha 2M, which serves as the proteinase trap, also nonspecifically binds cytokines. Arguments in support of the alternative model include the complex quaternary structure of alpha 2M, the known trapping mechanism by which alpha 2M interacts with proteinases, and the large number of structurally unrelated cytokines that have been reported to associate with alpha 2M (37).

In this study, we present evidence demonstrating, for the first time, that TGF-beta binding to alpha 2M is not dependent on alpha 2M quaternary structure and thus not dependent on the alpha 2M trap. Furthermore, we localize the TGF-beta -binding site to a single 20-kDa peptide that also contains the alpha 2M bait region. The 20-kDa peptide, when expressed as a GST fusion protein, binds both TGF-beta isoforms with equivalent or increased affinity, compared with intact alpha 2M. The peptide also neutralizes the activity of TGF-beta in endothelial cell proliferation assays and macrophage NO synthesis experiments. Thus, this 20-kDa peptide mimics the TGF-beta -regulatory activity of intact homotetrameric alpha 2M.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents and Proteins-- TGF-beta 2 was purchased from Genzyme (Cambridge, MA). TGF-beta 1 was from R & D Systems (Minneapolis, MN). Nerve growth factor-beta (NGF-beta ) was purified from male mouse submaxillary glands by the method of Darling and Shooter (38). Methylamine HCl, chloramine T, iodoacetamide (IAM), dithiothreitol (DTT), isopropylthio-beta -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.

alpha -Macroglobulins and Related Derivatives-- Human alpha 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. alpha 2M-MA was prepared by dialyzing human alpha 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 alpha 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 alpha 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 alpha 2M was separated from the monomers by FPLC on Superose-6. Monomeric alpha 2M, which is prepared as described, does not reassociate at 22 °C (44).

Preparation of Constructs Encoding GST-alpha 2M-Peptide Fusion Proteins-- The human alpha 2M cDNA in pAT153/PvuII/8 (pAT-alpha 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 right-arrow T). To generate a construct encoding GST-alpha 2M peptide fusion protein-1 (FP1), a fragment from pAT-alpha 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-alpha 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-alpha 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-alpha 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 alpha 2M cDNA inserts were in the correct orientation. Fig. 1 shows the relationship of the five peptides to the intact structure of alpha 2M.


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Fig. 1.   Schematic representation of the alpha 2M subunit and the alpha 2M peptides that were incorporated into GST fusion proteins. The amino acid numbering is based on the structure of the mature alpha 2M subunit.

Purification of GST-alpha 2M Peptide Fusion Proteins-- BL21 cells harboring pGEX-alpha 2M-peptide expression constructs were induced with 0.1 mM isopropylthio-beta -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.

Highly purified preparations of FP3 and FP4 were isolated in the absence of SDS by treating the original lysozyme extracts with 1.5% (w/v) Sarkosyl and 5 mM DTT. The FP3 and FP4, which solubilized in the Sarkosyl, were passed sequentially through 18- and 25-gauge needles and subjected to centrifugation at 12,000 × g. The supernatants, which contained the fusion proteins, were treated with Triton X-100 (2% v/v) to sequester the Sarkosyl (45) and then subjected to affinity chromatography on glutathione-Sepharose 4B. FP3 and FP4, which eluted from the column, were dialyzed against 1.5% Sarkosyl, 1 mM DTT and treated with IAM (5 mM) for 2 h at 25 °C to block free sulfhydryl groups. The final preparations were then dialyzed extensively against PBS.

Ligand Blotting-- Native alpha 2M, alpha 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-alpha 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 alpha 2M, alpha 2M-MA, MUG, and BSA were probed with 125I-TGF-beta 2 (20 pM), 125I-TGF-beta 1 (20 pM), or 125I-NGF-beta (50 pM) for 2 h at 25 °C; membranes with the five GST-alpha 2M peptide fusion proteins were probed with 125I-TGF-beta 2. The TGF-beta 1 and TGF-beta 2 were radioiodinated, to a specific activity of 100-200 µCi/µg, as described previously (46). NGF-beta 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-beta binding to FP3 is noncovalent and specific, membranes containing immobilized FP3 (0.5 µg) were incubated with 125I-TGF-beta 1 (0.25 nM) or 125 I-TGF-beta 2 (0.25 nM) in the presence of unlabeled TGF-beta 1 (200 nM), unlabeled TGF-beta 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-alpha 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-beta 2 to FP3 and FP4 as Determined by FPLC-- 125I-TGF-beta 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-beta 2-fusion protein complexes were separated from free 125I-TGF-beta 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-beta 2 was detected in elution fractions using a gamma -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-beta Binding to Immobilized alpha 2M-MA-- alpha 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 alpha 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 alpha 2M-MA. 125I-TGF-beta 1 or 125I-TGF-beta 2 (0.1 nM) was incubated with the immobilized alpha 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-beta , which was associated with the immobilized phase, was recovered in 0.1 M NaOH, 2% SDS and quantitated in a gamma -counter. Results were analyzed by plotting the specific binding of 125I-TGF-beta versus the log of the fusion protein concentration. In these experiments, the concentration of TGF-beta (TGF-beta 1 or TGF-beta 2) was at least 100-fold lower than the KD for TGF-beta -binding to immobilized alpha 2M-MA. Thus, TGF-beta -binding was linearly related to the free TGF-beta concentration ([beta F]), according to the following equation: B = (Bmax/KD)[beta F]. In the presence of a fusion protein (FP) that binds TGF-beta , the total concentration of TGF-beta ([beta T]) was related to [beta F], at equilibrium, as follows: [beta T] = [beta F](1 + [FP]/KI). If the fusion protein-TGF-beta complex did not bind to immobilized alpha 2M-MA, then TGF-beta -binding was reduced by 50% (the IC50) when [beta T]/[beta 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-beta 1 or TGF-beta 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-beta 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. alpha 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 alpha 2M increases RAW 264.7 cell NO synthesis by neutralizing endogenously produced TGF-beta (27). The alpha 2M-induced increase in RAW 264.7 cell NO synthesis is inhibited by the nitric-oxide synthase inhibitor, NG-monomethyl-L-arginine.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ligand Blot Analysis of 125I-TGF-beta Binding to alpha 2M-- Native alpha 2M, alpha 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 alpha 2M migrated as a single band with an apparent mass of 360 kDa, as expected; reduced alpha 2M migrated as a single major band with an apparent mass of 180 kDa (3). Methylamine treatment did not alter the mobility of alpha 2M (14, 15, 48).

125I-TGF-beta 2 bound to native alpha 2M and alpha 2M-MA, which were immobilized on PVDF membranes (Fig. 2). 125I-TGF-beta 2 binding was unchanged when the alpha 2M was treated with DTT or with IAM prior to electrophoresis. 125I-TGF-beta 2 also bound to BSA; however, this interaction was observed only after DTT treatment and was eliminated by treating the BSA with IAM. Thus, binding of 125I-TGF-beta 2 to reduced BSA probably involves free sulfhydryl groups that are not available in the native BSA structure. The ability of isolated alpha 2M subunits to bind 125I-TGF-beta 2, by an IAM-insensitive mechanism, suggests that the ligand blotting system accurately models the interaction of TGF-beta with nondenatured alpha 2M and that alpha 2M quaternary structure is not necessary for this interaction.


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Fig. 2.   Ligand blot analysis of 125I-cytokine binding to native alpha 2M, alpha 2M-MA, and BSA. Native alpha 2M, alpha 2M-MA, and BSA were denatured in 2% SDS, with or without 1 mM DTT, for 30 min at 37 °C. Some samples were subsequently treated with 5 mM IAM for 2 h at 25 °C. DTT- or IAM-treated samples are marked with a plus sign in the respective rows. All samples were subjected to SDS-PAGE and electrotransferred to PVDF membranes. The membranes were blocked and incubated with the indicated cytokines for 2 h at 25 °C. Cytokine binding was detected by PhosphorImager analysis.

To further assess the growth factor binding activity of isolated alpha 2M subunits in the ligand blotting system, studies were performed with 125I-TGF-beta 1 and 125I-NGF-beta . These two cytokines bind to nondenatured alpha 2M with similar affinity (31). As shown in Fig. 2, 125I-TGF-beta 1 and 125I-NGF-beta both bound to immobilized alpha 2M by an IAM-insensitive mechanism. Reductant-treated BSA also bound 125I-TGF-beta 1 and 125I-NGF-beta ; however, this interaction was eliminated when the BSA was treated with IAM.

Ligand Blot Analysis of the Binding of 125I-TGF-beta 2 to MUG-- MUG is a monomeric murine homologue of human alpha 2M. Although tetrameric murine alpha 2M, in its native form, and human alpha 2M bind TGF-beta 1 and TGF-beta 2 similarly, MUG does not bind either TGF-beta isoform with significant affinity (KD ~1.0 µM) (30). Thus, we compared the binding of 125I-TGF-beta 2 to human alpha 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-beta 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 alpha -macroglobulins. Apparently, MUG does not contain a cryptic TGF-beta -binding site that is exposed by SDS treatment.


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Fig. 3.   125I-TGF-beta 2 binding to alpha 2M and MUG, as determined by ligand blot analysis. Native alpha 2M and MUG were denatured in SDS. Samples that were DTT- or IAM-treated are designated by plus signs. All samples were subjected to SDS-PAGE and electrotransferred to PVDF membranes. The membranes were blocked and incubated with 125I-TGF-beta 2 for 2 h at 25 °C. 125I-TGF-beta 2-binding was detected by PhosphorImager analysis.

TGF-beta 2-Binding to GST-alpha 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-beta 2. Since all five fusion proteins were IAM-treated, free sulfhydryl groups in FP3 did not account for the 125I-TGF-beta 2 binding. FP1, FP2, FP4, FP5, and purified GST (not shown) did not bind 125I-TGF-beta 2.


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Fig. 4.   Binding of 125I-TGF-beta 2 to GST-alpha 2M-peptide fusion proteins. The five fusion proteins (FP1-FP5) were subjected to SDS-PAGE and electrotransferred to PVDF or nitrocellulose membranes. PVDF membranes were stained with Coomassie Blue. Western blot analysis was performed with an anti-GST IgG fraction of antiserum. Ligand blot analysis was performed with 125I-TGF-beta 2. After incubation for 2 h, 125I-TGF-beta 2-binding was detected by PhosphorImager analysis.

In separate ligand blotting experiments, affinity-purified FP3 and FP3 that was stored in SDS bound TGF-beta 2 equivalently (results not shown). Thus, the two preparations were interchangeable when analyzed by this method. In order to demonstrate that 125I-TGF-beta -binding to FP3 is noncovalent and specific, 125I-TGF-beta was incubated with PVDF-immobilized FP3 in the presence of excess solution phase FP3 or unlabeled TGF-beta . FP3 (1 µM) in solution inhibited the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to immobilized FP3 by 94 ± 3 and 92 ± 5%, respectively. Unlabeled TGF-beta 1 (0.2 µM) inhibited 125I-TGF-beta 1 binding to immobilized FP3 by 72 ± 8%; unlabeled TGF-beta 2 (0.2 µM) inhibited 125I-TGF-beta 2 binding to immobilized to FP3 by 90 ± 4%.

Binding of 125I-TGF-beta 2 to FP3 in Solution-- 125I-TGF-beta 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-beta 2 were separated by FPLC on Superose-12. We previously demonstrated that free TGF-beta 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-beta 2 was recovered with this fusion protein (n = 2). By contrast, only 6% of the TGF-beta 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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Fig. 5.   Binding of 125I-TGF-beta 2 to purified FP3 as determined by FPLC. FP3 and FP4 were incubated with 125I-TGF-beta 2 for 30 min at 37 °C and then subjected to FPLC on a Superose-12 column. Radioactivity recovery in each fraction is plotted as a percentage of the originally loaded radioactivity. The solid tracings show the absorbance of the eluate at 280 nm as a function of time.

125I-TGF-beta -Binding to Immobilized alpha 2M-MA-- 125I-TGF-beta 1 and 125I-TGF-beta 2 (0.1 nM) were incubated in separate alpha 2M-MA-coated microtiter wells for 1 h; 3.4 ± 0.3 fmol of 125I-TGF-beta 1 and 2.6 ± 0.2 fmol of 125I-TGF-beta 2 bound to the immobilized alpha 2M-MA. Unlabeled TGF-beta (0.2 µM) decreased the binding of 125I-TGF-beta by greater than 75%. 125I-TGF-beta recovery in the immobilized phase was decreased by greater than 95% when the wells were not precoated with alpha 2M-MA.

Various concentrations of FP3, FP4, or GST were added, with 125I-TGF-beta 1 or 125I-TGF-beta 2, to alpha 2M-MA-coated microtiter wells. FP3 inhibited the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to immobilized alpha 2M-MA, in a concentration-dependent manner (Fig. 6). Nearly complete inhibition was observed when the concentration of FP3 exceeded 100 nM; this result indicates that TGF-beta -FP3 complex does not bind to alpha 2M-MA. The IC50 was 33 ± 5 nM in experiments with TGF-beta 1 and 26 ± 6 nM in experiments with TGF-beta 2. If binding of a single FP3 to TGF-beta eliminates the ability of the TGF-beta to bind to alpha 2M-MA, then the IC50 provides an accurate estimate of the KD; if more than one FP3 must bind, then the KD is lower than the IC50. FP4 and GST did not inhibit the binding of 125I-TGF-beta 2 to immobilized alpha 2M-MA. GST also failed to inhibit the binding of 125I-TGF-beta 1 to immobilized alpha 2M-MA.


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Fig. 6.   Binding of 125I-TGF-beta to immobilized alpha 2M-MA in the presence of FP3 and FP4. In panel A, 125I-TGF-beta 2 was incubated in alpha 2M-MA-coated wells in the presence of increasing concentrations of affinity-purified FP3 (black-square), FP4 (bullet ), or GST (black-triangle) (the concentration of fusion protein was based on the molecular mass of the monomer). In panel B, 125I-TGF-beta 1 was incubated in alpha 2M-MA-coated wells in the presence of increasing concentrations of affinity-purified FP3 (black-square) or GST (black-triangle). Incubations were conducted for 1 h at 22 °C. After washing, 125I-TGF-beta , which was bound to the immobilized phase, was recovered in 0.1 M NaOH, 2% SDS. Radioactivity was quantitated in a gamma -counter (mean ± S.E., n = 4).

Table I summarizes studies comparing the activities of FP3, native alpha 2M, monomeric alpha 2M, alpha 2M-MA, and thrombospondin as solution phase inhibitors of the binding of TGF-beta to immobilized alpha 2M-MA. Some of the results were presented previously (31, 33); however, all of the IC50 values were determined by an identical method. The IC50 values determined for FP3, native alpha 2M, and alpha 2M-MA, in studies with TGF-beta 2, were all similar although the effective sequence in FP3 is contained in quadruplicate within the structure of intact, homotetrameric alpha 2M. The IC50 determined for TGF-beta 1 and FP3 was slightly lower than that determined with alpha 2M-MA and substantially lower than that determined with native alpha 2M. Interestingly, monomeric alpha 2M bound TGF-beta 1 with slightly increased affinity compared with native alpha 2M, although each mol of native alpha 2M contains 4 mol of monomer. This result may be explained if the affinity of TGF-beta 1 for its binding site in tetrameric alpha 2M is decreased compared with the affinity for the same site in monomeric alpha 2M and/or the number of available TGF-beta -binding sites within tetrameric alpha 2M is less than four.

                              
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Table I
Concentration of unlabeled competitor that decreases 125I-TGF-beta binding to immobilized alpha 2M-MA by 50% (IC50 values)
The IC50 values for alpha 2M, alpha 2M-MA, and thrombospondin were previously reported in Refs. 31 and 33. All values represent the mean ± S.E.; n = 4. 

FBHE Cell Proliferation Assays-- To determine whether FP3-binding inhibits TGF-beta activity, FBHE cell proliferation assays were performed. The cells were pulse-exposed to TGF-beta 1 or TGF-beta 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-beta 1 and TGF-beta 2, respectively. No change in TGF-beta activity was observed when FP4 was included in the medium. By contrast, FP3 nearly completely inhibited the activities of both TGF-beta 1 and TGF-beta 2, increasing [3H]thymidine incorporation to within 3 and 6% of the control values.

                              
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Table II
Effects of fusion proteins on TGF-beta activity in an endothelial cell proliferation assay
FBHE cells were pulse-exposed to TGF-beta (10 pM), FP3 (0.2 µM), FP4 (0.2 µM), or fusion protein + TGF-beta for 1 h. The fusion proteins were preincubated with TGF-beta for 15 min at 37 °C before addition to the FBHE cultures. After 30 h, 1 µCi/ml [3H]thymidine was added to the cultures for an additional 18 h. [3H]Thymidine incorporation was determined as a percentage of that observed in control cultures, which were not treated with TGF-beta or fusion protein.

Regulation of NO Synthesis by FP3 and FP4-- alpha 2M neutralizes TGF-beta , 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-beta , 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 alpha 2M-MA. We previously demonstrated that the increase in NO synthesis, which is induced by 280 nM alpha 2M-MA, is comparable with that observed with 10 ng/ml interferon-gamma (27). FP4 and purified GST (250 nM) did not increase nitrite production by the RAW 264.7 cells.


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Fig. 7.   NO synthesis by RAW 264.7 cells treated with FP3. RAW 264.7 cells were treated with alpha 2M-MA or FP3 in SFM. The control (C) was incubated in SFM (no alpha 2M-MA or FP3). After 24 h, nitrite levels in the conditioned media were measured. The presented results represent the mean ± S.E. (n = 4).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The TGF-beta 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-beta 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, beta -glycan, and endoglin. TGF-beta activity is also regulated by processes that alter delivery of the active cytokine to the cell surface. For example, TGF-beta is secreted as a large latent complex that includes latency-associated peptide and a second gene product, latent TGF-beta -binding protein (52-54). Conversion of latent TGF-beta into active 25-kDa homodimer requires dissociation of latency-associated peptide and latent TGF-beta -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-beta may bind to alpha 2M, once again forming a complex that is unavailable for receptor binding.

The fate of alpha 2M-associated TGF-beta depends on the alpha 2M conformation. Native alpha 2M, which is the predominant form of alpha 2M present in the plasma and probably in most extravascular microenvironments, binds TGF-beta reversibly and noncovalently (23, 31, 32). Thus, native alpha 2M may buffer tissues against rapid changes in TGF-beta levels by binding or slowly releasing the cytokine in response to the free TGF-beta concentration. Based on the KD value, we predict that approximately 95% of the TGF-beta 1 in plasma is alpha 2M-associated under equilibrium conditions, even though TGF-beta 1 binds to native alpha 2M with lower affinity than TGF-beta 2 (31). Conversion of alpha 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-beta is regulated. First, transformed alpha 2M has free Cys residues and thus undergoes thiol-disulfide exchange with TGF-beta (23, 31, 32), eliminating the potential for release of active cytokine. Second, alpha 2M-proteinase complexes bind to the endocytic receptor, LRP; bound TGF-beta is internalized with the alpha 2M-proteinase complex and probably delivered to lysosomes (22, 36).

The goal of the present investigation was to identify the binding site for TGF-beta in alpha 2M. Our original ligand blotting experiments, with human alpha 2M, demonstrated that intact quaternary structure is not necessary for TGF-beta binding. Treatment of alpha 2M with IAM did not inhibit TGF-beta -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-beta to purified MUG by ligand blotting, since nondenatured MUG, unlike human alpha 2M and tetrameric murine alpha 2M, does not bind TGF-beta with significant affinity (30). MUG bound only trace levels of TGF-beta in ligand blotting studies, supporting our hypothesis that ligand blotting provides a valid model of TGF-beta -alpha -macroglobulin interactions that occur under nondenaturing conditions. The inability of TGF-beta and NGF-beta to bind to reduced and alkylated BSA further supports the use of ligand blotting as a valid model system.

When the majority of the alpha 2M cDNA was expressed in a series of five GST fusion proteins, TGF-beta -binding was localized exclusively to FP3. The other four fusion proteins and purified GST did not bind TGF-beta . Selective binding of TGF-beta to affinity-purified FP3, and not to FP4, was demonstrated by FPLC and by radioligand-binding competition assay. FP3 was more effective than native alpha 2M or alpha 2M-MA at inhibiting TGF-beta 1 binding to immobilized alpha 2M-MA. This result is intriguing for at least three reasons. First, in comparing FP3 and intact alpha 2M, we used the concentrations of intact alpha 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 alpha 2M "subunit," then the difference between FP3 and intact alpha 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-beta -binding to immobilized alpha 2M-MA; otherwise, the KI is lower than the IC50. Although it is possible that two copies of FP3 or alpha 2M are required to neutralize TGF-beta , given the homodimeric structure of TGF-beta , this possibility is considered less likely with intact alpha 2M, due to its large size and complex structure, as discussed below. Also, as discussed below, our studies suggest that tetrameric alpha 2M may bind more than one molecule of TGF-beta . Finally, we cannot be certain that FP3 adopts a secondary and tertiary structure that is optimal for TGF-beta binding. Taken together, these results suggest that a specific sequence in FP3 binds TGF-beta with relatively high affinity. The equivalent sequence may be partially masked within intact alpha 2M, accounting for the observed decrease in TGF-beta binding affinity. The masking of the TGF-beta -binding site in intact alpha 2M may also explain why alpha 2M conformational change markedly alters TGF-beta binding affinity (31).

Human alpha 2M and bovine alpha 2M bind TGF-beta 2 with increased affinity compared with TGF-beta 1 (24, 31), explaining why TGF-beta 1 is preferentially active in certain cell culture assays that require serum-supplemented medium (24-26). Danielpour and Sporn (24) provided evidence that the alpha -macroglobulins from rabbit also preferentially bind TGF-beta 2. By contrast, murine alpha 2M binds TGF-beta 1 and TGF-beta 2 with equivalent affinity (30). In this study, we demonstrated that TGF-beta 1 and TGF-beta 2 bind to FP3 with equivalent affinity as well. This result suggests that the isoform specificity in TGF-beta binding to certain alpha -macroglobulins may be due to the ability of TGF-beta 2 to preferentially access the FP3-binding site in the intact alpha -macroglobulin. When the structural constraints of intact alpha 2M are eliminated, as in FP3, isoform specificity in TGF-beta binding is no longer observed. We do not understand why the binding site for TGF-beta 2 may be "less masked" in the structure of intact human alpha 2M compared with the binding site for TGF-beta 1; however, NMR and x-ray crystallography studies have demonstrated the presence of small differences in the overall shape and structure of TGF-beta 1 and TGF-beta 2 (59-61).

In addition to the TGF-beta -binding site, FP3 also contains the alpha 2M bait region. Models have been developed regarding the location of the bait region within the complex three-dimensional structure of alpha 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 alpha 2M resembles a hollow cylinder with a two-compartment central cavity. In alpha 2M-proteinase complexes, the proteinases occupy the central cavities. The bait regions are located within the central cavities, toward the center of the alpha 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-beta -binding site are equivalent or overlapping, then the TGF-beta -binding site may be accessible only from within the alpha 2M central cavity. TGF-beta -specific antibodies fail to recognize alpha 2M-associated TGF-beta (19, 24), supporting the hypothesis that TGF-beta occupies the central cavity; however, it is not clear whether the alpha 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-beta , within intact alpha 2M, remains unresolved. The bait region is known as an area of extreme sequence variability among alpha -macroglobulins from different species (5). Since TGF-beta -binding is conserved among many alpha -macroglobulins, with the exception of MUG (30, 31, 34), one can argue that the bait region and TGF-beta -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 alpha 2M has been estimated at 1:1 or 2:1 (19, 68). Our results suggest that the binding site contained within a single alpha 2M subunit may be sufficient to bind TGF-beta . Thus, an estimate of four cytokine-binding sites per alpha 2M does not seem unreasonable. Limitations in the number of cytokine-binding sites in intact alpha 2M may result from steric hindrance. If alpha 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 alpha 2M and TGF-beta requires that the cytokine engage two equivalent copies of FP3 on different subunits. KD values, determined by the alpha 2M-MA immobilization method and by our previously described BS3-cross-linking method (31, 33), assume a single cytokine-binding site per alpha 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 alpha 2M in biological assays.

In FBHE cell proliferation assays, we demonstrated that FP3 not only binds TGF-beta 1 and TGF-beta 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 alpha 2M-MA. Since we previously demonstrated that the induction of NO synthesis by alpha 2M is due to the neutralization of TGF-beta (27), we hypothesized that the increased potency of FP3 may be due to its increased binding affinity for TGF-beta 1. To test this hypothesis, we measured the secretion of TGF-beta 1 and TGF-beta 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-beta 1 were 2 and 10 pM, respectively. The concentrations of active and total TGF-beta 2 were 1 and 4 pM, respectively. The active TGF-beta 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-beta isoforms but higher levels of TGF-beta 1, supporting the hypothesis that the increased potency of FP3 reflects its increased capacity to neutralize TGF-beta 1.

In summary, we have identified a single peptide from the structure of alpha 2M that contains the binding site for TGF-beta 1 and TGF-beta 2. The high affinity of FP3 for both TGF-beta isoforms and the substantial potency of FP3 in two TGF-beta neutralization assays suggests that the TGF-beta -binding sequence may be partially masked in intact alpha 2M. Like TGF-beta 1 and TGF-beta 2, NGF-beta bound to dissociated alpha 2M subunits, suggesting that intact quaternary structure and the resulting alpha 2M central cavity or trap is not necessary. However, at this time, we have not determined whether the NGF-beta -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 alpha 2M and for further refinement of the TGF-beta binding sequence.

    FOOTNOTES

* 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: alpha 2M, alpha 2-macroglobulin; alpha 2M-MA, alpha 2M-methylamine; MUG, murinoglobulin; TGF-beta , transforming growth factor-beta ; NGF-beta , nerve growth factor-beta ; 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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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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