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Originally published In Press as doi:10.1074/jbc.M502068200 on May 24, 2005

J. Biol. Chem., Vol. 280, Issue 29, 27044-27055, July 22, 2005
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Characterization of the Interaction between Tumor Necrosis Factor-stimulated Gene-6 and Heparin

IMPLICATIONS FOR THE INHIBITION OF PLASMIN IN EXTRACELLULAR MATRIX MICROENVIRONMENTS*

David J. Mahoney{ddagger}§, Barbara Mulloy¶, Mark J. Forster¶, Charles D. Blundell||**, Eric Fries{ddagger}{ddagger}, Caroline M Milner, Supported by a Nuffield Foundation Oliver Bird Fellowship (RHE/00045/G){ddagger}§§§, and Anthony J. Day{ddagger}¶¶

From the {ddagger}Medical Research Council Immunochemistry Unit and the ||Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, Hertfordshire, United Kingdom, and the {ddagger}{ddagger}Department of Medical Biochemistry and Microbiology, Uppsala University, S-751 23 Uppsala, Sweden

Received for publication, February 23, 2005 , and in revised form, May 2, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-{alpha}-inhibitor (I{alpha}I). Experiments using the purified components of I{alpha}I indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6·I{alpha}I complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TSG-61 (the secreted product of tumor necrosis factor-stimulated gene-6), a protein composed mainly of contiguous Link and CUB modules, is not constitutively expressed in adult tissues but is up-regulated in many different inflammatory diseases that often involve remodeling of the extracellular matrix (ECM) (1). These include rheumatoid arthritis and osteoarthritis (2, 3), Kawasaki disease (4), systemic lupus erythematosus (5), and asthma.2 TSG-6 is also expressed in normal physiological processes involving ECM reorganization, notably following blood vessel wall injury (6), during ovulation (6-10), and in cervical ripening (11). TSG-6 binds to several components of the ECM through its Link module domain; i.e. the glycosaminoglycans (GAGs) hyaluronan (HA) (12-17) and chondroitin-4-sulfate (13), as well as the G1 domain of aggrecan (18) and pentraxin-3 (19, 20). Interactions between TSG-6 and the serine protease inhibitor inter-{alpha}-inhibitor (I{alpha}I) have also been described (21-27). I{alpha}I is a proteoglycan and consists of three polypeptides (heavy chain 1 (HC1), heavy chain 2 (HC2) and bikunin), covalently linked by a chondroitin sulfate moiety, which originates from Ser-10 of bikunin (28). There is evidence that TSG-6 mediates the cross-linking of HA chains in the ECM by acting as an essential cofactor in the formation of HA·HC covalent complexes (23, 25) and that this occurs via covalent TSG-6·HC intermediates (20, 26, 27). We have shown previously that TSG-6 can also interact with I{alpha}I in a non-covalent fashion to potentiate its anti-plasmin effect (see Ref. 29), however the molecular basis of this interaction has not been characterized in detail.

Increases in plasmin activity have been implicated in many disease pathologies involving ECM reorganization, including tumorigenesis and angiogenesis (30), atherosclerotic plaque formation (31), rheumatoid arthritis (32), multiple sclerosis (33), and bacterial infection (34), as well as in processes such as follicle rupture during ovulation (35). Plasmin acts by activating matrix metalloproteinases and by liberating growth factors and cytokines sequestered within the ECM. Studies using mouse models of arthritis have shown that TSG-6 has a chondroprotective effect, with reduced loss of cartilage proteoglycan and less accumulation of metalloproteinase and aggrecanase-generated aggrecan fragments; this is consistent with the suggestion that TSG-6 is exerting its protective effect through inhibition of plasmin (36-39). Indeed, in the context of proteoglycan-induced arthritis, tsg-6-/- mice show increased plasmin activity in inflamed paw joints compared with wild-type mice (40). Furthermore, in mice with antigen-induced arthritis, constitutive expression of TSG-6 in cartilage resulted in a less severe disease phenotype with delayed onset of symptoms relative to controls (39).

Heparin, a heavily-sulfated polysaccharide consisting of alternating residues of uronic acid (either {alpha}-L-iduronic acid or {beta}-D-glucuronic acid) and {alpha}-D-glucosamine connected via 1->4-glycosidic linkages, is best known as a modulator of protease function through its interaction with serine protease inhibitors. I{alpha}I is also a heparin-binding protein (41), and therefore it is possible that this GAG may have an effect on its anti-plasmin activity. We have reported previously that heparin is unable to compete for HA binding to the Link module of TSG-6 (13), and hence we concluded that this domain does not interact with heparin. However, given that the CUB module in other proteins (e.g. the spermadhesins) binds to heparin (42, 43), it was thought possible that the TSG-6 CUB module could mediate such an interaction. Therefore, we have revisited the question of whether heparin/heparan sulfate binds to TSG-6 (comparing its interaction with the full-length protein and Link module) and investigated the effect of heparin on the ability of TSG-6 to potentiate the inhibitory activity of I{alpha}I toward plasmin.

In this study we demonstrate, using microtiter plate assays and isothermal titration calorimetry (ITC), an interaction between TSG-6 and heparin/heparan sulfate via its Link module domain; the position of the heparin-binding site was predicted by computational docking and was then verified by site-directed mutagenesis. Importantly, the binding of heparin augments the potentiation by TSG-6 of the anti-plasmin activity of I{alpha}I. We propose that this inhibition of plasmin arises through the formation of a ternary complex in which heparin may enhance the interaction between the Link module of TSG-6 and the bikunin chain of I{alpha}I. We also describe a role for heparin in inhibiting subsequent interactions between TSG-6 and HA but have confirmed that heparin is not an effective inhibitor when both these GAGs are present. These results suggest that there may be partitioning of TSG-6 into different functional pools depending on the composition of GAGs in different tissue microenvironments.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Wild-type and Mutant Proteins—The Link module from human TSG-6 (Link_TSG6; residues 36-133 in the preprotein (44)) and Link_TSG6 mutants H4K (where His-4 of Link_TSG6 was replaced with Lys), K11Q, Y12F, Y59F, F70V, K72A, and Y78F were expressed in Escherichia coli, purified to homogeneity and characterized by mass spectrometry and NMR as described previously (15, 29). The same methodology was used to make six additional single-site and six multiple-site Link_TSG6 mutants of residues predicted to be involved in heparin binding (i.e. K20A, K34A, K41A, K54A, R56A, R84A, K34A/K54A, K20A/K34A/K41A, K20A/K41A/K54A, K20A/K34A/K54A, K34A/K41A/K54A, and K20A/K34A/K41A/K54A; see "Results"). These new mutants were analyzed by electrospray ionization mass spectrometry and by one-dimensional NMR to determine if they were folded, as described previously (15). Each protein had an experimental mass within 3.0 Da of the theoretical value and was stored lyophilized at -20 °C.

The full-length human TSG-6 protein (allelic variant TSG-6Q with Gln at amino acid position 144 in the pre-protein) was expressed in Drosophila Schneider 2 cells and purified by ion-exchange chromatography and reverse-phase high-performance liquid chromatography as before (22). The concentrations of wild-type/mutant Link_TSG6 and full-length protein were determined by amino acid analysis as described previously (15). I{alpha}I was purified from human serum as in a previous study (45), and the individual heavy chains (HC1 and HC2) were then isolated and their concentrations determined, as described previously (46). Human bikunin was prepared as before (47).

Binding of TSG-6 Proteins to Immobilized Heparin and Heparan Sulfate—The heparin/heparan sulfate-binding activities of TSG-6 and Link_TSG6 were compared using a colorimetric enzyme-linked immunosorbent assay that measures the binding of protein to heparin/heparan sulfate immobilized non-covalently on the surface of a microtiter plate plasma polymerized with allylamine (supplied by Plasso Technology Ltd., Sheffield, UK) as described in detail in Ref. 48. Briefly, the plates were incubated with heparin (either high molecular weight heparin as reported in the 4th international standard (49, 50) or low molecular weight heparin (H-3400; Sigma)) overnight at room temperature, using 0-5 µg/well in 200 µl of phosphate-buffered saline. Alternatively, heparan sulfate from bovine kidney (H-9637; Sigma) or HSI and HSII preparations isolated from a GAG-rich pig mucosal fraction by ion exchange chromatography (51) were used instead. Plates were washed in standard assay buffer (SAB-1; 50 mM sodium acetate, 100 mM NaCl, 0.2% (v/v) Tween 20, pH 6.0) and blocked with 1% (w/v) bovine serum albumin in SAB-1 for 90 min at 37 °C as described before (15). Wells were rewashed and incubated with 8 pmol/well Link_TSG6 or TSG-6 in SAB-1 for 4 h. Bound protein was detected by the addition of 1.25 µg/well Q75 monoclonal antibody (52) in 200 µl of SAB-1 for 45 min, followed by 200 µl of alkaline phosphatase-conjugated goat anti-rat IgG (Sigma), diluted 1:2000 in SAB-1, for an additional 45 min. All microtiter plate assays were developed by adding 200 µl/well of 1 mg/ml disodium p-nitrophenyl phosphate in 0.05 M Tris-HCl, 0.1 M NaCl, 5 mM MgCl2, pH 9.3. The absorbance values at 405 nm were measured after 5 min and corrected against blank wells (i.e. those that contained no heparin).

The heparin-binding activities of Link_TSG6 mutants were compared with that of the wild-type Link module using the enzyme-linked immunosorbent assay described above except that the proteins were incubated on plates coated at a heparin concentration of 1 µg/well. Protein binding was detected using either Q75 or A38; rat monoclonal antibodies, which recognize different epitopes in the TSG-6 Link module (52).

Isothermal Titration Calorimetry—The interaction between Link_TSG6 and a defined octasaccharide of heparin (denoted here as Hp8) was investigated on a MicroCal VP-ITC instrument at 25 °C in 5 mM sodium Mes, 95 mM NaCl, pH 6.0, as previously described for HA (15, 16, 29, 53); heparin oligomers were prepared from bovine lung heparin (material remaining from the second international standard (54)), purified using high performance gel permeation chromatography and the concentrations determined by measuring the area under the chromatography peaks compared with a standard curve prepared using heparin of known concentration as described previously (55). Hp8 (ranging from 0.26 to 0.59 mM) was added, in injections 28 x 5 µl, into protein solution at 0.015-0.087 mM. Data were fitted to a one-site model by non-linear least squares regression with the Origin software package, after subtracting the heats resulting from the addition of oligomer into buffer alone, as described previously (14, 16).

Molecular Modeling—The position of the heparin-binding site on the TSG-6 Link module was predicted using Autodock version 2.4 (56), implemented as described previously (55, 57). This program allows for flexibility in the ligand structure but uses a rigid-body protein approximation to speed up the calculation. Docking was performed with pentasaccharide (5-mer) or endecasaccharide (11-mer) models of heparin (consisting, respectively, of three or six GlcN2S6S residues separated by IdoA2S residues) and Link_TSG6 in either its free or HA-bound conformations (pdb accession codes 1o7b [PDB] and 1o7c, respectively (16)); calculations were performed with each of the 20 lowest energy NMR structures for both the "free" (closed, pdb 1o7b [PDB] ) and "bound" (open, pdb 1o7c [PDB] ) Link module. To take account, at least in part, of variability in the IdoA ring geometry, two different 5-mer molecules were used in which all the IdoA2S residues adopted either the 1C4 or 2S0 ring form. The model ligands had fixed glycosidic torsion angles based on those reported for the structure of heparin in solution (58); for the pentasaccharide models, rotation around all other exocylic bonds was allowed. No bond rotation was allowed for the endecasaccharide model, in which IdoA2S residues took the 1C4 conformation. Partial atomic charges for the heparin monosaccharides were derived from ab initio quantum chemistry calculations using a HF/6-31G* basis set within the Jaguar program (Schrodinger Inc.). Automation of the docking calculation was achieved using Perl code to generate the Autodock input files, run the calculation, and analyze the output. The ten lowest energy coordinate sets were calculated for each of the Link module structures analyzed.

HA Binding Assay—The effect of heparin on the interaction of HA with TSG-6 or Link_TSG6 was investigated using our standard assay, which measures the binding of biotinylated-HA (bHA) to protein-coated microtiter plates (see Refs. 15 and 22). Maxisorp plates (Nunc) were coated with 8 pmol/well TSG-6 or 25 pmol/well Link_TSG6, then washed and blocked with 1% (w/v) bovine serum albumin in SAB-2 (50 mM sodium acetate, 100 mM NaCl, 0.05% (v/v) Tween 20, pH 6.0); as described before (22), these coating conditions give rise to essentially identical levels of HA binding. Plates were then incubated with bHA (12.5 ng/well), in the absence or presence of low molecular weight heparin (0-500 ng/well), for 4 h in SAB-2 at room temperature. Following washing with SAB-2, bound bHA was detected using ExtrAvidin alkaline phosphatase as described before (15), where the absorbance at 405 nm was determined after 10 min. Alternatively, plates were preincubated with heparin (0-500 ng/well) for 2 h and washed in SAB-2 prior to addition of the bHA and then developed in an identical manner.

Interaction of I{alpha}I with Heparin and TSG-6 —I{alpha}I, or its individual components (i.e. bikunin, HC1 or HC2), were coated onto microtiter plates at 0-20 pmol/well, which were then washed in SAB-2 and blocked as before (15). Biotinylated-heparin (b-heparin, made from high molecular weight heparin as described previously for bHA (15)) was incubated (19.8 ng/well in 200 µl of SAB-2) in the absence or presence of unlabeled heparin for 4 h, and bound b-heparin was detected with ExtrAvidin alkaline phosphatase as in Ref. 15; plates were read after a 10-min development time.

The binding of mono-biotinylated-Link_TSG6 (bA-Link_TSG6 (13)) and TSG-6 to plates coated with intact I{alpha}I or its protein chains (2 pmol/well) was also investigated; bA-Link_TSG6 and TSG-6 were incubated for 4 h in SAB-1 at 2 pmol/well and 5 pmol/well, respectively. The amount of bound bA-Link_TSG6 was determined with ExtrAvidin alkaline phosphatase as before, whereas TSG-6Q was detected by incubation with a rabbit anti-human polyclonal antiserum against TSG-6 (11) diluted 1:4000 in SAB-1, followed by incubation with an alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson Immuno-Research, 1:2000 in SAB-1) for 45 min each; the absorbance values at 405 nm were determined after 8- and 5-min development times, respectively. As before, competition experiments were set up to determine whether the interactions observed were specific.

Single-site Link_TSG6 mutants were subsequently used to localize the Link module binding surface on bikunin. Mutant Link_TSG6 (at 2 pmol/well in SAB-1) was incubated for 4 h on plates coated with 2 pmol/well bikunin. Bound protein was determined using Q75 monoclonal antibody as before, after a 12-min development time.

Analysis of Plasmin Inhibition—The effects of low molecular weight heparin and rooster comb HA on the ability of Link_TSG6 to potentiate the anti-plasmin effect of I{alpha}I were determined using the assay described before (29). I{alpha}I (48 nM) was preincubated with Link_TSG6 (740 nM) in 10 mM sodium acetate, 150 mM NaCl, 0.02% (v/v) Tween 20, pH 6.0, in the absence or presence of 1.78 µg of GAG (i.e. an ~10-fold molar excess (6 µM) of heparin over Link_TSG6) for 30 min at 37 °C. Anti-plasmin activity in all cases was assessed by the addition of plasmin and chromozym-PL (giving final concentrations of 3.4 nM plasmin, 197 µM chromozym-PL, 24 nM I{alpha}I, 370 nM Link_TSG6, and, where appropriate, 3 µM heparin). The absorbance values at 405 nm were measured after 25 min, and protease activity was expressed as a percentage of plasmin activity in the absence of I{alpha}I and Link_TSG6. The pH dependence of GAG-augmented potentiation of anti-protease activity was determined by repeating these experiments at pH 6.25, 6.5, 6.75, or 7.0 (using sodium acetate buffer as before) as well as at pH 7.4 (in 10 mM HEPES-HCl, 150 mM NaCl, 0.02% (v/v) Tween 20).

The anti-plasmin activity of the isolated bikunin chain (with HC1 and HC2 as controls; all at 48 nM) was measured in the absence and presence of Link_TSG6 at pH 6.0 and 7.4 as described above. The potentiation activity of certain Link_TSG6 mutants was then determined; I{alpha}I (48 nM) was preincubated with the single-site mutants (740 nM), prior to the measurement of plasmin activity at pH 6.0 as before. The effect of mutation on the ability of heparin to augment the Link_TSG6-mediated potentiation of the anti-plasmin activity of I{alpha}I was also investigated: I{alpha}I (at 48 nm) was preincubated with single-site mutants (740 nM) in the presence of 6 µM heparin, prior to the determination of plasmin activity at pH 6.0 as before.

The amount of heparin required for maximal augmentation of anti-plasmin activity at pH 6.0 was determined by co-incubating different molar ratios of the GAG (0.027-3.56 µg) with Link_TSG6 (740 nM) and I{alpha}I or bikunin (at 48 nM), prior to the addition of plasmin and chromozym-PL and development of the assay as above. In a further assay constant amounts of heparin (6 µM and 1.78 µg) and I{alpha}I (48 nM) were incubated with various concentrations of Link_TSG6 (0-740 nM) prior to the determination of plasmin activity at pH 6.0 as before.



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  		FIG. 1.
Comparison of the heparin- and heparan sulfate-binding activities of TSG-6 and Link_TSG6. The interactions of TSG-6 (closed symbols) and Link_TSG6 (open symbols) with high molecular weight heparin (a) or heparan sulfate (HS) (b) preparations (HSI, HSII, and HS from bovine kidney (BK)) immobilized on microtiter plates (plasma polymerized with allylamine) were determined by enzyme-linked immunosorbent assay as described under "Experimental Procedures." All the values are plotted as mean absorbances (n = 4) ± S.E.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Heparin/Heparan Sulfate-binding Site on the Link Module of TSG-6 —The interaction of TSG-6 with heparin/heparan sulfate was revisited using an assay where heparin or heparan sulfate is non-covalently immobilized on a microtiter plate plasma polymerized with allylamine (see Ref. 48); this new assay allows the use of the microtiter plate format to investigate heparin/heparan sulfate-protein interactions. The binding of Link_TSG6 or recombinant full-length TSG-6 (Q allotype; the most common variant in the Caucasian population (22)) was assessed with a Link module-specific monoclonal antibody. As can be seen from Fig. 1a, Link_TSG6 and TSG-6 (both at 8 pmol/well) bind to high molecular mass heparin (~12 kDa) in an essentially identical dose-dependent manner; similar results were seen with low (~3 kDa) molecular mass preparations of heparin (data not shown). This indicates that TSG-6 is able to interact with heparin and that the binding site is within its Link module domain. Consistent with this, assays that measured the interaction of b-heparin with plates coated with Link_TSG6 or TSG-6Q (at concentrations that bind equivalent amounts of HA (22)) revealed similar levels of heparin binding (data not shown). Furthermore, the isolated Link module and the full-length protein were both able to interact with three different types of heparan sulfate (Fig. 1b), including the HSI and HSII preparations, where the former has a lower level of sulfation than the latter (51). This reveals that, in addition to heparin, the TSG-6 Link module is able to interact with heparan sulfate, potentially binding to a wide variety of heparan sulfate sequences. In this regard, heparin was used in all subsequent experiments, because this is less heterogeneous than heparan sulfate, and represents a suitable model for the highly sulfated S-domains present in heparan sulfate.



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  		FIG. 2.
Analysis of the interaction of Link_TSG6 with a heparin octasaccharide by ITC. A representative titration plot for the binding of Hp8 to Link_TSG6 (C value = 17.8) derived from the integrated raw data after subtraction of heats of dilution for the injectant (Hp8). The solid line represents the least squares best fit to the data for a single binding-site model. The derived stoichiometry (N), binding constant (Kb), and enthalpy ({Delta}bH), averaged from five independent experiments, are shown in Table I.

 
The interaction of heparin with the Link module was further investigated in solution by ITC, where a defined octasaccharide of heparin (Hp8) was injected into Link_TSG6 in the sample cell as described previously for HA (14-16, 53); the oligosaccharides were prepared from a sample of bovine lung heparin containing >90% (w/w) of the trisulfated disaccharide IdoA2S-GlcNS6S (54), making them unusually homogeneous in structure. Fig. 2 shows a representative titration plot for one of five titrations summarized in Table I. From Table I, it can be seen that the binding of Link_TSG6 to Hp8 has an ~6-fold higher affinity than its interaction with an 8-mer of HA (see Ref. 53) determined under identical conditions (i.e. 5 mM Mes, 95 mM NaCl, pH 6.0). For both GAGs the enthalpy is the major factor driving the interaction, however, in the case of heparin the entropy makes a larger contribution to the binding energy. Unlike HA8, which binds to Link_TSG6 in a 1:1 ratio (14, 16, 53) the stoichiometry for the interaction with Hp8 was determined to be 0.46 ± 0.06 (Table I). This value is close to 0.5 and therefore it seems likely that two Link_TSG6 molecules bind to an 8-mer of heparin to form a protein dimer. A titration with Hp6 also gave an N value of 0.45, where the binding in this case was estimated to be 5-fold weaker than for Hp8 (data not shown). ITC experiments with Hp4 (at 25 °C) gave no measurable heats indicating that if this oligosaccharide binds at all it will be with a significantly lower Kb.


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  		TABLE I
Binding constants and thermodynamic parameters for the interactions of Link_TSG6 with heparin and hyaluronan ITC was used to investigate the binding of octasaccharides of heparin (Hp8) and hyaluronan (HA8) to Link_TSG6 in 5 mM Mes, 95 mM NaCl, pH 6.0. In the case of heparin the experimentally determined values (i.e., stoichiometry (N), Kb, and {Delta}bH) are given as the mean of five separate experiments (±S.E.), where three different 8-mer stocks were used for which the concentration was determined independently; for these experiments C-values range from 7.5 to 24.2. The HA values were taken from Ref. 53.

 
Modeling the Heparin-binding Site on TSG-6 —The heparin-binding site on the TSG-6 Link module was predicted by systematic docking calculations using coordinates for Link_TSG6 in both its free (closed) and HA-bound (open) conformations (16) with 5- and 11-mer heparin models; these theoretical oligomers, which are composed of IdoA2S-GlcNS6S disaccharides, are very similar in structure to the heparin used in the ITC experiments described above. The "closed" and "open" states of the Link module gave essentially identical results, predicting the heparin-interaction surface to be distinct from the HA-binding groove across both families of NMR structures. Docking of the pentasaccharide models, for which the sulfate substituent groups were allowed to rotate freely, identified three basic amino acids, Lys-34, Lys-54, and Arg-56, as interacting with the Link module in all the low energy predicted complexes. Predictions for both 1C4 and 2S0 IdoA2S forms of the pentasaccharide were similar in geometry and energies (data not shown).



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  		FIG. 3.
Prediction of the heparin-binding site on the TSG-6 Link module. The middle panel shows the ten lowest energy coordinate sets resulting from modeling the heparin 11-mer (green sticks with sulfur atoms represented as small yellow spheres) onto the Link module (space-filling) in its closed conformation; this is illustrated for docking calculations performed with the lowest energy Link_TSG6 NMR structure (see Ref. 16). The heparin 11-mers lie in two possible orientations (denoted H1 and H2, respectively) that are predicted to involve three common basic amino acids (dark blue). Orientation H1 (bottom left), which was predicted in 80% of the models, utilizes additional residues (light blue) that extend on either side of this central patch, whereas H2 (bottom right) is orthogonally arranged to include Lys72 (green) in its predicted binding site. In the bottom panel a single 11-mer is shown for each of the orientations, where the sulfurs are represented as atomic spheres. Both orientations are distinct from, and non-overlapping with, the position of the HA-binding groove (residues in red (15, 16)).

 
As can be seen from Fig. 3 two possible orientations of the heparin endecasaccharide ligand were identified (H1 and H2) that are arranged orthogonally to one another on a face of the Link module adjacent to, but not overlapping with, the HA-binding site. Approximately 80% of 11-mers docked onto the Link module (i.e. 400 endecamers on 40 Link_TSG6 structures) along the H1 axis. Both binding orientations include the basic amino acids (i.e. Lys-34, Lys-54, and Arg-56 shown in dark blue in Fig. 3), predicted to make contact with the pentasaccharides. Additional electrostatic contacts are predicted from Lys-20, Lys-41, and Arg-84 (light blue) in orientation H1 and from Lys-72 (green) in H2.

Mapping the Heparin-binding Site by Mutagenesis—We have demonstrated previously that 5 residues of Link_TSG6 (Lys-11, Tyr-12, Tyr-59, Phe-70, and Tyr-78), which are clustered on one face of the Link module (16), have an important role in the interaction with HA; e.g. the mutants K11Q, Y12F, Y59F, F70V, and Y78F have between 10- and 100-fold lower HA-binding affinity compared with wild-type protein (15, 29). From Fig. 4 it can be seen that these mutants have no impairment in their ability to interact with immobilized heparin, consistent with the prediction from molecular modeling that the heparin- and HA-binding sites are distinct and non-overlapping. Furthermore, the mutation of His-4, which is on the opposite face of the Link module to the predicted heparin-binding site, to lysine (i.e. H4K), has no effect on heparin-binding activity.

To further test the docking prediction, twelve single and multiple site Link_TSG6 mutants were generated (see "Experimental Procedures"), in addition to the K72A mutant that had been made previously (15). One-dimensional NMR revealed that the new lysine single site mutants (i.e. K20A, K34A, K41A, and K54A) all had spectra essentially identical to wild-type protein (data not shown) and were therefore correctly folded; as described in detail before (15) we are able to classify Link_TSG6 mutants as having a wild-type fold, a perturbed fold, or being unfolded on the basis of their NMR spectrum. For example, mutation of Arg-56 or Arg-84 to alanine perturbed the Link module fold, and these mutants were not investigated further. Of the multiple site mutants only K34A/K54A and K20A/K34A/K41A gave spectra corresponding to the wild-type structure, the others (i.e. K20A/K34A/K41A, K20A/K41A/K54A, K20A/K34A/K54A, and K20A/K34A/K41A/K54A) being unfolded/perturbed (data not shown). Mass spectrometry revealed that the mutants with wild-type folds all had experimental masses within 1.5 Da of their theoretical masses.

As can be seen from Fig. 4, mutations of Lys-34 and Lys-54 (i.e. residues predicted to make up the central portion of the heparin-binding site) cause statistically significant reductions in the ability of Link_TSG6 to bind to immobilized heparin; K34A, K54A, and K34A/K54A have between 45 and 69% of wild-type functional activity as detected by the Q75 monoclonal antibody. The K20A and K41A mutants also have a significant impairment in their binding activity (55 and 58% of wild-type, respectively), whereas mutation of Lys-72 to alanine did not have any affect on binding.

These data, as well as demonstrating the involvement of Lys-20, Lys-34, Lys-41, and Lys-54 in heparin binding, also indicate that this GAG is likely to interact with the TSG-6 Link module via the H1 rather than the H2 orientation. Consistent with this, the triple mutant K20A/K34A/K41A has a heparin-binding activity that is 10-fold lower than the wild-type protein (Fig. 4). From Fig. 5 it is apparent that Lys-20, Lys-34, Lys-41, and Lys-54 are arranged in a line across the Link module surface providing good evidence that heparin does indeed bind along the H1 axis. There is clearly no overlap between the heparin- and HA-interaction surfaces, such that these GAGs, if they were to bind simultaneously, would be arranged orthogonally to one another. In this regard, microtiter plate assays indicate WT levels of bHA binding for each of the single and multiple site mutants that have impaired heparin binding (data not shown).

Effect of Heparin on HA Binding—Competition experiments, with both the isolated Link module and full-length TSG-6 (Fig. 6), confirmed a previously reported observation (13) that bHA binding is not effectively competed by heparin when these GAGs are co-incubated with the immobilized protein (IC50 = ~500 ng of heparin for Link_TSG6, i.e. 40 times the amount of bHA used in the assay). However preincubation of heparin, with either immobilized TSG-6 or Link_TSG6, resulted in an increase of ~30-fold in the ability of heparin to compete for bHA binding (i.e. it is an equimolar competitor under these conditions). Similar experiments showed that heparin was an effective competitor for biotinylated-C4S binding to Link_TSG6/TSG-6-coated plates, regardless of whether it was co- or preincubated (data not shown), indicating that C4S may interact at the heparin-binding site.

These data show that the TSG-6 Link module is unable to bind HA and heparin simultaneously, despite the fact that their binding sites are non-overlapping. One possible explanation for this is that heparin stabilizes the Link module in its closed confirmation (as shown in Fig. 5) in which the HA-binding residues are less accessible; interaction of Link_TSG6 with HA requires a conformational change in the Link module that opens a groove on the module surface (16). Conversely, HA binding, which stabilizes the protein in its open state (16), may affect the relative orientation of the amino acids involved in binding to heparin. It should be noted, however, that no differences were seen in the docking predictions for Link_TSG6 in its open and closed states. An alternative explanation for the inhibition of heparin binding to TSG-6 by HA is that the interaction with HA is cooperative and leads to fiber formation (as suggested by our recent observations (59)), and that such interactions occlude the heparin binding surface. It will be necessary to perform further structural studies to provide a clear answer to this question.



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  		FIG. 4.
Comparison of heparin-binding activities of wild-type and mutant Link_TSG6. The binding of wild-type (WT) and mutant Link_TSG6 proteins to high molecular weight heparin immobilized on allylamine plates was detected using the Q75 monoclonal antibody; the double and triple-site mutants K34A/K54A and K20A/K34A/K54A are denoted by D and T, respectively. Similar results were seen when plates were coated with a low molecular weight preparation of heparin (data not shown). Values are plotted as the mean percentage of maximal absorbance for WT protein (n = 8) ± S.E.; ***, p < 0.001 compared with WT control. Western blot analysis indicated that K20A and K20A/K34A/K41A (T) were poorly recognized by the Q75 antibody compared with the wild-type and other mutant proteins (data not shown) and, therefore, this could account for the apparent reduction in heparin-binding activity of these mutants. However, assays done with an alternative detection antibody (A38), which reacts with a different epitope in the Link module and recognized K20A, the triple mutant and wild-type protein equally on Western blots, gave essentially identical results to those reported here (data not shown) for all mutants except Y78F, which has been found previously not to be recognized by this antibody (51).

 
Heparin Binding to TSG-6 Affects Its Potentiation of the Anti-plasmin Activity of I{alpha}I—Given the above finding that heparin binds to the TSG-6 Link module and our previous observation that this domain potentiates the anti-plasmin activity of I{alpha}I (29), we investigated the effect of heparin on this process. As can be seen from Fig. 7a, heparin was found to enhance the Link_TSG6-mediated potentiation of the anti-plasmin activity of I{alpha}I in a pH-dependent manner. This effect of heparin was maximal at pH 6.0 and decreased as the pH was raised to 7.4; addition of HA (or C4S) had no effect at either pH extreme (data not shown). Importantly, heparin did not enhance the inhibition of plasmin by I{alpha}I in the absence of TSG-6 across this range of pH values. At pH 7.0 and 7.4, although Link_TSG6 was able to potentiate the anti-plasmin activity of I{alpha}I as described before (29), no significant increase in plasmin inhibition was seen in the presence of heparin (data not shown). Furthermore, mutants of Link_TSG6 with reduced heparin-binding function showed WT potentiation activities in the absence of heparin (Fig. 7b, black bars). However, in the presence of heparin (Fig. 7b, gray bars) there was generally a reduction in the heparin-mediated augmentation of the potentiation activity (e.g. the K34A, K54A, K34A/K54A, and K20A/K34A/K41A mutants all had significantly reduced activities compared with wild-type in assays containing heparin). It should be noted that the correlation between the effect of a particular mutation on heparin binding to free Link_TSG6 and its effect in the plasmin assay was less clear than might have been anticipated. This apparent discrepancy may arise due to the complexity of the plasmin assay, i.e. that a multimolecular complex is likely to be formed between TSG-6, heparin, and I{alpha}I (see below). In such a complex a composite heparin-binding site may be generated that can compensate for the loss of particular ionic interactions between the Link module and the GAG. For example, it is well established that heparin binds to both protein components of a fibroblast growth factor (FGF)·FGF receptor complex (60). Furthermore, the precise heparan sulfate sequence that is optimal for eliciting a biological response is determined by the particular FGF and FGF receptor subtypes involved and does not necessarily correspond to the sequence that exhibits maximal binding to the FGF alone (61).

TSG-6 Interacts with the Bikunin Chain of I{alpha}I—To further understand the mechanism of potentiation of the anti-plasmin activity of I{alpha}I by TSG-6 and its augmentation by heparin a number of questions were investigated (e.g. where in I{alpha}I does TSG-6 and heparin bind?). Firstly, it was shown that Link_TSG6 only interacts with the bikunin chain of I{alpha}I, with no binding observed for either HC1 or HC2 (see Fig. 8a); the binding of Link_TSG6 to I{alpha}I was not competed by C4S (data not shown). Similar results were obtained for the full-length TSG-6, where this gave a comparable level of binding to bikunin as seen for Link_TSG6 (Fig. 8a); some binding of TSG-6 to immobilized HC2 was evident, but this was not reduced by the inclusion of competing HC2 (in solution) indicating that this interaction is likely to be nonspecific in nature (data not shown). Furthermore, in plasmin assays with the individual I{alpha}I chains, Link_TSG6 had essentially the same potentiation effect on bikunin as on intact I{alpha}I, whereas in samples containing HC1 or HC2 there was no inhibition of protease activity (see Fig. 8b). These results demonstrate that TSG-6 potentiates the anti-plasmin activity of I{alpha}I via a protein-protein interaction between its Link module and bikunin.

Given that I{alpha}I has been shown previously to interact with heparin (41), a property used in its purification from sera (62), it is possible that this interaction could play a role in the augmentation of its anti-plasmin activity. However, as described above, heparin had no effect on plasmin inhibition by I{alpha}I in the absence of TSG-6. Consistent with this, although heparin was shown to bind to both HCs of I{alpha}I, it did not interact with bikunin (Fig. 9). Therefore, it seems clear that heparin binding to the Link module of TSG-6 is responsible for the augmentation of its potentiation of the anti-plasmin activity of I{alpha}I.

Mapping the Bikunin-binding Site on the TSG-6 Link Module—As described above we have shown that both heparin and bikunin interact with the TSG-6 Link module. To determine the position of the bikunin-binding site, various single-site Link_TSG6 mutants were tested for both their bikunin-binding properties and "potentiation" activities. Four of these mutants, shown previously to have a large reduction in their HA-binding activities (i.e. K11Q, Y12F, Y59F, and Y78F (15, 29)), also had significantly reduced ability to interact with bikunin (Fig. 10a); F70V that has a 10-fold lower binding affinity for HA than the wild-type protein (29) had unaltered binding to bikunin. The single-site Link_TSG6 mutants that have impaired heparin-binding properties (i.e. K20A, K34A, K41A, and K54A (see Fig. 4)) all showed wild-type binding to bikunin. These data indicate that bikunin binds to the Link module at a site overlapping, but not identical to, its HA binding surface and that this interaction does not involve the residues that mediate binding to heparin. Consistent with this, F70V had essentially a wild-type potentiation activity, whereas K11Q, Y12F, and Y59F show an almost complete loss of function, and Y78F had a modest but significant reduction (Fig. 10b); the potentiation activities determined here for Y12F, F70V, and Y78F (i.e. 11, 93, and 81% of wild-type, respectively) are similar to those we have reported previously for these mutants (i.e. 18, 94, and 70%, respectively (29)). Therefore, overall there is a good correlation between the effect of mutation on bikunin binding and potentiation activity. The only exception to this is the Y78F mutant that, while retaining ~80% of wild-type potentiation function, does not appear to bind bikunin alone (Fig. 10); mutation of Tyr-78 to valine also effectively abolishes binding to bikunin, but the mutant retains 52% of wild-type activity in the plasmin assay (data not shown). At present we have no explanation for this apparent discrepancy, however, it seems clear that these Tyr-78 mutants can interact with bikunin in the presence of plasmin.



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  		FIG. 5.
The heparin- and HA-binding sites on the TSG-6 Link module are non-overlapping. The Link module is illustrated in its closed and open conformations, i.e. those determined previously in the absence and presence of HA, respectively (16). In the left-hand panel, a low energy prediction for a heparin 11-mer docked onto the closed Link_TSG6 structure is shown, where the heparin could interact with the basic amino acids Lys-20, Lys-34, Lys-41, and Lys-54 (colored as in Fig. 3 and labeled on the open conformer for clarity); these residues have been demonstrated by mutagenesis to be involved in heparin binding. On the right is shown a model of an HA 8-mer docked into the Link module in its open conformation (as described (17)), where the residues colored red mediate this interaction. The heparin- and HA-binding sites are orthogonal and non-overlapping. Given that the binding of Link_TSG6 to heparin inhibits its subsequent interaction with HA, and that HA binding requires a conformation change in the Link module that opens a groove on its surface (16), it is possible that its association with heparin stabilizes the closed conformer of the protein making the key HA-binding residues inaccessible (i.e. heparin inhibits HA binding via an allosteric mechanism).

 



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  		FIG. 6.
The effect of heparin in modulating the interaction of bHA with Link_TSG6 and TSG-6. Heparin (low molecular weight) was either preincubated (p) with immobilized protein prior to addition of bHA or added to wells at the same time as bHA and co-incubated (c); closed and open symbols for Link_TSG6 and TSG-6, respectively. Mean values (n = 8) are plotted as a percentage of binding in the absence of heparin ± S.E.

 
Mutagenesis of any of the four lysine residues shown to be involved in heparin binding (Lys-20, Lys-34, Lys-41, and Lys-54; see Fig. 4) has no significant effect on the ability of Link_TSG6 to potentiate the anti-plasmin activity of I{alpha}I (Fig. 10b). Interestingly, these mutations do have a slight effect on the interaction of the Link module with bikunin (72-85% of WT binding; Fig. 10a). It is possible therefore that mutating these amino acids to alanine may destabilize the bikunin binding surface, perhaps through allosteric cross-talk between the heparin and bikunin/HA-binding sites. Alternatively, these residues may take part in bikunin-induced Link module-Link module self-association, which may be necessary for potentiation of I{alpha}I function (see below). Whatever the reasons behind this observation, in the presence of plasmin (i.e. on its binding to bikunin) these mutants (like Y78F) are highly active.

Role of Heparin in Augmenting the Potentiation Activity of TSG-6 —As can be seen from Fig. 11a, the enhancement of Link_TSG6-mediated potentiation of the anti-plasmin activity of both I{alpha}I and bikunin by heparin is dose-dependent. Close to maximum inhibition (i.e. approximately a 2- or 4-fold increase of plasmin inhibition by bikunin or I{alpha}I, respectively, in the absence of heparin) is seen when the heparin is at a final concentration of 3 µM (i.e. 1.78 µg); i.e. the amount of heparin used throughout the rest of this study.

In earlier studies the molar ratio of TSG-6/Link_TSG6 to I{alpha}I used in the plasmin assays was 15:1 (29, 63); as noted previously this ratio gives rise to maximal inhibition of plasmin by wild-type Link_TSG6 (29). Here we examined the effect of varying the ratio of Link_TSG6:I{alpha}I in the presence of an optimal amount of heparin (i.e. 3 µM final concentration). From Fig. 11b it is apparent that in the presence of heparin, close to maximal inhibition of plasmin is observed when these proteins are present at a 4:1 ratio.

Given that heparin binding to Link_TSG6 is likely to cause dimerization of the Link module (see Table I and "Results" above), it seems plausible that four molecules of TSG-6 (i.e. two dimers) could bind to each molecule of I{alpha}I. In this regard, the bikunin chain, with which Link_TSG6 interacts (Fig. 8), is composed of two contiguous and closely packed kunitz domains, where only the first of these has an accessible protease recognition site (64); the second kunitz domain is obstructed by the first. Therefore, an attractive model of TSG-6/heparin-mediated potentiation of the anti-plasmin activity of I{alpha}I would be for the binding of two heparin-stabilized TSG-6 dimers to bikunin (perhaps, one per kunitz domain) to cause a separation of the two kunitz domains such that second protease inhibitor region becomes less sterically hindered (see Fig. 12). In the context of such a complex, bikunin may interact directly with heparin; in the bikunin crystal structure two sulfate ions were found to be coordinated to the protein (64). Furthermore, it is possible that TSG-6 enhances the interaction of plasmin with bikunin, perhaps via a direct binding site for plasmin on its Link module; in this regard, we have found that TSG-6 is a substrate for plasmin cleavage.3 Clearly, further structural and functional studies are required to determine the exact nature of the multimolecular complex formed between heparin, TSG-6, and I{alpha}I and how this interacts with plasmin.



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  		FIG. 7.
The effect of heparin on the Link_TSG6-mediated potentiation of the anti-plasmin activity of I{alpha}I. a, plasmin activity was determined by a chromogenic assay at pH 6.0, 6.25, 6.5, 6.75, 7.0, and 7.4, in the presence of I{alpha}I and Link_TSG6, with (gray) or without (black) excess low molecular weight heparin (3 µM final concentration). All values are expressed as a mean percentage (n = 4) of plasmin activity alone at the appropriate pH ± S.E.; ***, p < 0.001 compared with value at pH 7.4 without heparin. The reduction with increasing pH in the ability of heparin to augment Link_TSG6 potentiation is mirrored by the pH dependence of the Link_TSG6/heparin interaction; i.e. the binding of b-heparin to Link-TSG6-coated plates decreases significantly as the pH is raised from 6.0 to 7.4 (data not shown). b, The effect of mutation on the Link_TSG6-mediated potentiation of the anti-plasmin activity of I{alpha}I at pH 6.0, in the presence (gray) or absence (black) of 1.78 µg of heparin. All values are expressed as a mean percentage (n = 4) of plasmin activity alone ± S.E.; ***, p < 0.001; *, p < 0.05 compared with WT potentiation activity in the presence of heparin.

 



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  		FIG. 8.
The enhanced anti-plasmin activity of I{alpha}I in the presence of TSG-6 arises through an interaction between the Link module and the bikunin chain. a, the interactions of Link_TSG6 (black) and full-length TSG-6 (gray) with the individual protein subunits of I{alpha}I at pH 6.0. Component chains of I{alpha}I were adsorbed onto microtiter surfaces in 20 mM Na2CO3, pH 9.6, and the binding determined by enzyme-linked immunosorbent assays using bA-Link_TSG6 or a TSG-6 specific polyclonal antiserum, respectively. Mean absorbance values (n = 8) are expressed as a percentage of I{alpha}I binding (for Link_TSG6 or TSG-6 as appropriate) ± S.E. b, the anti-plasmin activities of the individual protein chains of I{alpha}I were measured in the presence of Link_TSG6 using chromozym-PL as substrate at pH 6.0. Mean values (n = 8) are expressed as a percentage of plasmin activity in the absence of I{alpha}I/TSG-6 ± S.E.; ***, p < 0.001 compared with the activity of plasmin alone.

 



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  		FIG. 9.
I{alpha}I interacts with heparin via its heavy chains. The protein subunits of I{alpha}I were immobilized on microtiter plates at various concentrations and the binding of b-heparin determined at pH 6.0 (i.e. the pH at which there is maximal augmentation of the anti-plasmin activity of I{alpha}I in the presence of Link_TSG6). I{alpha}I = open circle, bikunin = open square, HC1 = closed circle, HC2 = closed square. Data are shown as mean absorbances (n = 8) for the interactions of the proteins with heparin (±S.E.).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we show that the Link module of TSG-6, in addition to interacting with HA, also contains a binding site for heparin/heparan sulfate. The similar binding properties of the full-length TSG-6 and Link_TSG6 proteins indicate that only the Link module contains a heparin-binding site (i.e. the CUB module does not interact with heparin); this may also be the case for heparan sulfate, but further work is required to confirm this. Computational docking calculations were used to predict the position where heparin binds to the Link module, as a model for the highly sulfated S-domains present in heparan sulfate, and these predictions were then tested by site-directed mutagenesis. This led to the identification of four basic amino acids involved in the binding of this GAG, where the heparin- and HA-binding sites are distinct from each other with no obvious overlap; the heparin-binding residues are brought together from different parts of the Link module sequence. It should be noted that chondroitin 4-sulfate and dermatan sulfate, which we have found to also bind TSG-6 (13),4 are both likely to interact with the Link module via its heparin binding surface.

We estimate that the heparin-binding site on the Link module should be able to accommodate about six monosaccharide residues in a heparin chain. This is consistent with our ITC studies, which indicate that an octamer is likely to be the minimum size of heparin oligosaccharide that can bind with maximum affinity to Link_TSG6 (i.e. accounting for end effects). The binding constant for the interaction with Hp8 is ~5 times greater than the corresponding value obtained with an 8-mer of HA (53) determined under identical conditions; HA8 is the minimum even-sized (testicular hyaluronidase-derived) HA oligomer that interacts maximally with Link_TSG6 (16). This higher affinity is not due to an increase in the enthalpy of the interaction, which is slightly lower than the corresponding value for HA binding (53), but rather to a more positive entropic contribution. Interestingly, favorable entropies have been reported previously for other heparin-protein associations (65, 66), and it has been suggested that this is likely to be due to the polyelectrolyte effect (see Ref. 67).



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  		FIG. 10.
The bikunin-binding site on Link_TSG6 overlaps the HA-interaction surface. a, the binding of Link_TSG6 mutants to immobilized bikunin at pH 6.0 was determined using the Q75 monoclonal antibody; mean values (n = 12) are shown as a percentage of wild-type ± S.E. b, the effect of Link_TSG6 mutants on potentiating the anti-plasmin activity of I{alpha}I (in the absence of heparin). Mean absorbance values at pH 6.0 (n = 12) are plotted as a percentage of control plasmin activity ± S.E.; ***, p < 0.001; **, p < 0.01 compared with the activity of the wild-type protein.

 
The reduction in functional activity following mutation of individual heparin-binding residues in the Link module is significantly less than has been observed when HA-binding amino acids are mutated (15, 29); i.e. between 45 and 69% compared with 1-10% of wild-type activity, respectively. This suggests that, in the case of heparin, binding is mediated through an extensive network of electrostatic interactions between the protein and the polysaccharide, where the loss of any one of these can be compensated for by additional contacts; the docking calculations predicted 6 basic amino acids in the H1 orientation as being involved in heparin binding; this includes Arg-56 and Arg-84, which did not give rise to folded mutants. A large positively charged interaction surface of this type, and the need to mutate several amino acids to cause a large reduction in binding (as we have found here for Link_TSG6), are characteristic of many heparin/heparan sulfate-protein interactions (see Ref. 67). This degeneracy could allow TSG-6 to interact with heparin and heparan sulfate sequences with a wide range of sulfation patterns. In this regard the Link module domain and full-length TSG-6 protein were demonstrated here to bind to three different preparations of heparan sulfate, where HSI is known to contain a low to moderate degree of sulfation (51).



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  		FIG. 11.
Dose dependences of heparin and Link_TSG6 on potentiation of the anti-plasmin activity of I{alpha}I. a, the dose-dependent effect of low molecular weight heparin (0-3.56 µg/well) on the anti-plasmin activities of I{alpha}I (closed circle) or bikunin (open square), in the presence of Link_TSG6 (at molar ratios of I{alpha}I/bikunin:Link_TSG6 of 1:15); plasmin assays were carried out at pH 6.0 with chromozym-PL as substrate as described under "Experimental Procedures." Mean absorbance values (n = 4) are plotted as a percentage of control plasmin activity (i.e. with no I{alpha}I or TSG-6) ± S.E. b, the effect of changing the molar ratio of TSG-6:I{alpha}I on the potentiation of the anti-plasmin activity of I{alpha}I in the presence of 1.78 µg of heparin (3 µM final concentration) at pH 6.0; I{alpha}I was at the standard final concentration of 24 nM. Mean absorbance values (n = 4) are plotted as a percentage of control plasmin activity ± S.E.

 
The interactions of TSG-6 with heparin/heparan sulfate, HA (and other GAGs), may have important implications for its functions in particular tissue microenvironments. In this regard, TSG-6 molecules could be partitioned into different functional pools depending on the composition of GAGs present in ECM or on cell surfaces. For example, here we have shown that the association of TSG-6 with heparin can inhibit subsequent HA binding, and therefore could negatively regulate HA-dependent functions.

Several functions of TSG-6 have been attributed recently to its HA-binding activity; e.g. its involvement in the expansion of the cumulus cell-oocyte complex (COC) during ovulation (see Ref. 20). COC expansion, which is necessary for successful ovulation and fertilization in vivo, is driven by the synthesis of HA and requires the stabilization of a HA-rich ECM surrounding the oocyte. In this regard, TSG-6 may contribute to stabilization via its formation of a multimeric complex with the protein PTX3, a decamer composed of 10 identical protomer subunits, in which the HA-interaction surface on the Link module is still accessible (19, 20); i.e. TSG-6/PTX3 could form a node cross-linking multiple HA chains. It should be noted that both PTX3 (19) and tsg-6 (23) null mice are severely sub-fertile due to a failure in COC expansion. The TSG-6-HA interaction is also likely to have a role in the regulation of lymphocyte adhesion during inflammation, through a modulation of the HA-binding affinity of CD44 (59); preincubation of HA with TSG-6 can enhance or induce CD44 ability to bind HA, probably via the formation of cross-linked HA fibers that can cause receptor clustering and activation. Thus, changes in the presentation of heparan sulfate sequences on proteoglycans, or the release of heparin due to mast cell degeneration, might be expected to modulate such HA-dependent processes.



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  		FIG. 12.
A hypothetical model for the potentiation of the anti-plasmin activity of bikunin by Link_TSG6 and heparin. ITC experiments indicate that the binding of heparin to Link_TSG6 causes dimerization of the protein. Such Link_TSG6/heparin dimmers may then bind to each of the two kunitz-type serine protease inhibitory domains of bikunin (labeled I and II). This could lead to a change in the relative orientations of kunitz domains I and II making the protease-binding sites (denoted as black ovals) more accessible, and thus causing an increase in the anti-plasmin activity of bikunin.

 
I{alpha}I has also been found to play a vital role in COC expansion via the formation of HC·HA complexes in which its heavy chains become covalently linked to HA, leading to a stabilization of the cumulus matrix (68, 69); bikunin-deficient mice, which are unable to synthesize the intact I{alpha}I proteoglycan, are female infertile. TSG-6 has been shown to catalyze the production of HC·HA complexes through its formation of covalent TSG-6·HC intermediates (20, 23, 25-27); it is believed that this occurs via two transesterification reactions where a heavy chain is first transferred from the chondroitin sulfate moiety of I{alpha}I onto TSG-6 and then from TSG-6 onto HA. The finding here that TSG-6 binds to bikunin, but not the I{alpha}I heavy chains, indicates that the association of the Link module with bikunin could constitute the initial interaction in the formation of TSG-6·HC complexes; this will require investigation. Although the molecular details of HA binding to TSG-6 have been well characterized (15-17), it is not clear at present whether the interaction between its Link module and HA is involved in the formation of HC·HA complexes. If this were the case, then it might be anticipated that heparin binding to TSG-6 would inhibit this process, however, we have found that this GAG does not affect either TSG-6·HC complex formation or HA transfer.5

The binding of heparin to TSG-6 does have a significant effect on its ability to potentiate the anti-plasmin activity of I{alpha}I. Although I{alpha}I alone only causes 5-8% inhibition, in the presence of TSG-6 this increases to ~40% (29, 63). We have found here that heparin binding to the TSG-6 Link module increases this anti-plasmin activity still further, up to ~80% at pH 6.0, and with a statistically significant augmentation seen between pH 6.0 and 6.75. Clearly this could be of functional importance at inflammatory sites where TSG-6 is expressed (see Ref. 1), I{alpha}I is present (because it can ingress into tissue spaces from serum (27, 69)), and where the production of heparin or heparan sulfate proteoglycans are up-regulated. Furthermore, inflammation is often associated with acidosis, and therefore this process could be regulated by the pH of the tissue microenvironment; i.e. in a similar fashion to our previous proposal for the regulation of the interactions of TSG-6 with HA and aggrecan in the context of inflamed cartilage (18). As noted in the introduction, tsg-6-/- mice have increased plasmin activity in their inflamed paw joints compared with wild-type controls in an experimental model of rheumatoid arthritis (40). In addition, the level of expression of the heparan sulfate proteoglycan perlecan has been found to be increased in human cartilage during osteoarthritis and may represent an attempt by the tissue to stabilize the ECM (70, 71).

As described above, the severe female infertility seen in mice due to deletion of either the bikunin (68) or tsg-6 (23) genes is thought to result from the lack of HC·HA complexes, correlating with the loss of the TSG-6·HC intermediates required in its formation (20, 23, 26, 27). The results described here suggest that non-covalent TSG-6·I{alpha}I complexes might also form in the ovarian follicle and could play a role in the regulation of plasmin. Indeed, bikunin is released on TSG-6·HC complex formation (26), due to the breakdown of a by-product of the reaction (27), and could therefore associate with free TSG-6; this is also relevant to inflammatory conditions where HC·HA and TSG-6·HC complexes have been detected (e.g. in synovial fluids of arthritis patients (see Refs. 27 and 69)). In support of this, plasmin activity has been observed to be up-regulated during ovulation (see Ref. 35) and at other sites of TSG-6 expression, such as in the cartilage of rheumatoid arthritis patients (see Ref. 1). For example, urokinase and tissue-type plasminogen activators are secreted by preovulatory ovarian follicles in response to gonadotrophins, leading to the activation of metalloproteinases that play an important role in the ECM remodeling involved in corpus luteum formation (72, 73). Clearly these processes require stringent regulation, and it is possible that TSG-6 and I{alpha}I could play a role in this. It should be noted that anti-coagulant heparan sulfate proteoglycans are hormonally induced in preovulatory follicles and co-localize with heparin-binding protease inhibitors (74-76). Interestingly, anti-coagulant heparan sulfate (74, 76) and TSG-6 (8) have very similar localization patterns in the antral granulosa cells of preovulatory ovarian follicles of rodents.

Finally, TSG-6 expression can be regulated by p53 (77), which might allow TSG-6 to play a role in tumorigenesis through its association with bikunin. In this regard, the relatively poor inhibition of plasmin by bikunin in solution is increased on the surface of cancer cells (78) that often express altered profiles of heparan sulfate proteoglycans (see Ref. 79). However, it remains to be seen whether TSG-6 is involved in this.

The finding reported here that the binding of heparin to TSG-6 augments its potentiation of the anti-plasmin activity of I{alpha}I could have widespread biological implications, especially in the context of inflammation as described above. It is also possible that these interactions may affect other activities of bikunin (e.g. modulation of cell growth and inhibition of cellular calcium uptake; see Ref. 80), and further work will be necessary to investigate this possibility. Furthermore, structural and functional studies are now required to determine the molecular details of the non-covalent ternary complex formed between heparin, TSG-6, and I{alpha}I, and to identify the heparan sulfate sequences that can augment TSG-6 function.


   FOOTNOTES
 
* This work was supported in part by the United Kingdom Medical Research Council and Arthritis Research Campaign (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by the ARC (Grants M0621, M0625, 16119, and 16539). Back

** Supported by an ARC PhD studentship (Grant D0569). Back

§§ To whom correspondence may be addressed. Tel.: 44-1865-275348; Fax: 44-1865-275729; E-mail: caroline.milner{at}bioch.ox.ac.uk. ¶¶ To whom correspondence may be addressed. Tel.: 44-1865-275349; Fax: 44-1865-275729; E-mail: tony.day{at}bioch.ox.ac.uk.

1 The abbreviations used are: TSG-6, tumor necrosis factor-stimulated gene-6; bHA, biotinylated hyaluronan; b-heparin, biotinylated heparin; bA-Link_TSG6, mono-biotinylated Link_TSG6; C4S, chondroitin-4-sulfate; COC, cumulus cell oocyte complex; ECM, extracellular matrix; HA, hyaluronan; GAG, glycosaminoglycan; I{alpha}I, inter-{alpha}-inhibitor; HC, heavy chain; ITC, isothermal titration calorimetry; Link_TSG6, the Link module of human TSG-6; SAB, standard assay buffer; WT, wild-type protein; Mes, 4-morpholineethanesulfonic acid; FGF, fibroblast growth factor. Back

2 R. Forteza, S. Casalino-Matsuda, M. E. Monzon-Medina, M. S. Rugg, C. M. Milner, and A. J. Day, manuscript in preparation. Back

3 D. J. Mahoney, M. S. Rugg, and A. J. Day, unpublished data. Back

4 D. J. Mahoney, B. Mulloy, and A. J. Day, manuscript in preparation. Back

5 M. S. Rugg, E. Fries, C. M. Milner, and A. J. Day, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Marilyn S. Rugg for the preparation of recombinant full-length TSG-6 protein and Plasso Technology Ltd. (Sheffield, UK) for supplying microtiter plates plasma polymerized with allylamine.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides
Glycobiology, October 1, 2007; 17(10): 1094 - 1103.
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 J. Biol. Chem.Home page
C. D. Blundell, D. J. Mahoney, M. R. Cordell, A. Almond, J. D. Kahmann, A. Perczel, J. D. Taylor, I. D. Campbell, and A. J. Day
Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan
J. Biol. Chem., April 27, 2007; 282(17): 12976 - 12988.
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 Am. J. Respir. Cell Mol. Bio.Home page
R. Forteza, S. M. Casalino-Matsuda, M. E. Monzon, E. Fries, M. S. Rugg, C. M. Milner, and A. J. Day
TSG-6 Potentiates the Antitissue Kallikrein Activity of Inter-{alpha}-inhibitor through Bikunin Release
Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 20 - 31.
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S. J. Clark, V. A. Higman, B. Mulloy, S. J. Perkins, S. M. Lea, R. B. Sim, and A. J. Day
His-384 Allotypic Variant of Factor H Associated with Age-related Macular Degeneration Has Different Heparin Binding Properties from the Non-disease-associated Form
J. Biol. Chem., August 25, 2006; 281(34): 24713 - 24720.
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 BrainHome page
A. Al'Qteishat, J. Gaffney, J. Krupinski, F. Rubio, D. West, S. Kumar, P. Kumar, N. Mitsios, and M. Slevin
Changes in hyaluronan production and metabolism following ischaemic stroke in man
Brain, August 1, 2006; 129(8): 2158 - 2176.
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 J. Biol. Chem.Home page
S. A. Kuznetsova, A. J. Day, D. J. Mahoney, M. S. Rugg, D. F. Mosher, and D. D. Roberts
The N-terminal Module of Thrombospondin-1 Interacts with the Link Domain of TSG-6 and Enhances Its Covalent Association with the Heavy Chains of Inter-{alpha}-trypsin Inhibitor
J. Biol. Chem., September 2, 2005; 280(35): 30899 - 30908.
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