The TSG-6 and I alpha I interaction promotes a transesterification cleaving the protein-glycosaminoglycan-protein (PGP) cross-link.

During co-incubation of human inter-alpha-inhibitor (IalphaI) and human tumor necrosis factor-stimulated gene 6 protein (TSG-6) SDS-stable interactions are formed between the two proteins. We have analyzed the products of this reaction and characterized the mechanism of complex formation. Following the incubation seven new bands not previously identified were apparent in SDS-PAGE. Three of these bands did not contain TSG-6, including heavy chain (HC)1.bikunin, HC2.bikunin, and free bikunin. In addition high molecular weight complexes composed of the same components as I alpha I, including HC1, HC2, and bikunin, were formed. The formation of these complexes was prevented by the addition of hyaluronan. The cross-links stabilizing these complexes displaying properties similar to the protein-glycosaminoglycan-protein (PGP) cross-link. The TSG-6-containing SDS-stable complexes were composed of HC1.TSG-6 or HC2.TSG-6 exclusively. Both glycosylated and non-glycosylated TSG-6 participated in the complex formation. The HC.TSG-6 cross-links were different from the PGP cross-link and were determined to be ester bonds between the alpha-carbonyl of the C-terminal Asp of the heavy chain and most likely a hydroxyl group containing the TSG-6 residue. The mechanism involved cleaving the PGP cross-link of I alpha I during a transesterification reaction. A TSG-6 hydroxyl group reacts with the ester bond between the alpha-carbonyl of the C-terminal Asp residues of HC1 or HC2 and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. An intermediate is formed resulting in a partitioning of the reaction between HC(1 or 2).TSG-6 complexes and transfer of HC(1 or 2) to the chondroitin via competing pathways.

Bikunin inhibits several serine proteases, but more effective inhibitors exist, and the physiological relevance of the protease inhibitor activity remains unclear (3). Three combinations of bikunin and HCs have been identified in human plasma, including (i) inter-␣-inhibitor (I␣I) composed of heavy chain 1 (HC1), heavy chain 2 (HC2), and bikunin, (ii) pre-␣-inhibitor (P␣I) composed of heavy chain 3 (HC3) and bikunin, and (iii) HC2⅐bikunin composed of HC2 and bikunin (1,4). One of the characteristics of bikunin is its involvement in novel crosslinks with the HCs. The molecular structure of the cross-link has previously been determined (5)(6)(7). It is mediated by a chondroitin-4-sulfate (CS) chain that originates from a typical O-glycosidic link on Ser-10 of bikunin. The C-terminal Asp residues of the HCs are esterified via the ␣-carbon to C-6 of an internal N-acetylgalactosamine of the CS chain. HC1 and HC2 are most likely in close proximity on the 12-to 18-disaccharidelong CS chain, and HC1 is attached further away from bikunin than HC2 (8). The cross-link is referred to as a protein-glycosaminoglycan-protein (PGP) cross-link (5) and is formed intracellularly ϳ30 min after the onset of the biosynthesis (4). The bikunin proteins are present in high concentrations in the blood (9) and in the extracellular matrix (ECM) (10 -12). In the ECM I␣I interacts with tumor necrosis factor-stimulated gene 6 protein (TSG-6).
TSG-6 is a secreted protein of ϳ35 kDa produced during inflammation and inflammation-like processes (13). The mRNA encoding for the protein was originally identified following tumor necrosis factor treatment of human diploid FS-4 fibroblasts (14,15). Like the members of the bikunin proteins, TSG-6 is thought to play a vital role in the ECM in general and particularly during cumulus cell⅐oocyte complex stabilization (13,16). The mammalian ovulation is accompanied by the permeabilization of the blood/follicle barrier, allowing ingress of blood proteins (17). During this process hyaluronan (HA), I␣I, and TSG-6 interact and stabilize the expansion of the cumulus cell⅐oocyte complex. The importance of these interactions has been emphasized in animal models where the bikunin or the TSG-6 genes have been knocked out. Both the bikuninand TSG-6-deficient mice were unable to assemble the ECM surrounding the oocyte causing infertility (11,16).
The interactions between TSG-6 and I␣I have been studied in vitro. After a brief incubation of purified human I␣I and human TSG-6 an apparent covalent complex between components of I␣I and TSG-6 is formed (18,19). It has been suggested that this complex is composed of HC2, bikunin, and TSG-6 (18).
In addition the TSG-6 and I␣I complex formation has been observed in vivo underscoring the biological importance of the cross-linking (20,21).
In this study we show that the interaction between I␣I and TSG-6 leads to the formation of seven distinct reaction products not previously characterized. These include two high molecular weight (HMW) I␣I species, HC1⅐TSG-6, HC2⅐TSG-6, HC1⅐bikunin, HC2⅐bikunin complexes, and free bikunin. The data suggest that the PGP ester bond between the ␣-carbonyl of the C-terminal Asp and carbon-6 of an internal N-acetylgalactosamine in the CS chain is cleaved during a transesterification reaction. An intermediate is formed, and a partitioning occurs between HC(1 or 2)⅐TSG-6 complex formation or transfer of HCs to the CS through competing pathways. , hyaluronic acid sodium salt from human umbilical cord, 1,10-phenanthroline, and polyethylene glycol 8000 were obtained from Sigma. Immobilon-P membrane was from Millipore. Biodyne B membrane (0.45 m) was obtained from Pall, Gelman Laboratory, and microspin filters (0.2 m) were from Lida. ECL Western blotting detection reagents, 5-ml Blue-Sepharose High Performance columns, 5-ml HiTrap Q High Performance columns, and S-300 HR column material were from Amersham Biosciences. Zorbax SB C18, 3.5-m reverse phase column material was from Agilent. PicoFrit columns (75-m ID) were from New Objective. Mass Spectrometry grade Trypsin was from Promega/Ramcon. Human plasma was obtained from Statens Serum Institut, Copenhagen, Denmark. I␣I and TSG-6 were purified as previously described (1,15,18). Antiserum against TSG-6 was produced as described before (15,18). Both TSG-6 antiserum and purified TSG-6 protein were kindly provided by Dr. Hans-Georg Wisniewski, New York University Medical Center, Department of Microbiology, New York.

Materials-Bovine
SDS-Polyacrylamide Gel Electrophoresis-Samples were boiled in SDS sample buffer in the presence of 10 -50 mM dithiothreitol. SDS-PAGE was performed in 5-15% gradient gels or in 7.5% uniform gels (10 cm ϫ 10 cm ϫ 0.15 cm) using the glycine/2-amino-2-methyl-1,3propandiol/HCl system described previously (22). The gels were stained for protein in the conventional way using Coomassie Blue or by the trypsin inhibition counter-staining technique (TIC staining) that specifically visualizes trypsin inhibitory activity (1,23).
Immunoblotting-Samples were resolved in 7.5% or 5-15% SDSpolyacrylamide gels and electrophoretically transferred to an Immobilon-P membrane (polyvinylidene difluoride membrane) (24). The membranes were blocked for 1 h at 25°C in 20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6 (TBS-T) containing 5% skim milk powder. The blots were incubated over night at 4°C in TBS-T containing 1% milk and rabbit antiserum against TSG-6. The next day the blots were washed in TBS-T and incubated with peroxidase-conjugated goat antirabbit IgG (Sigma) for 1 h. Following additional TBS-T washing, TSG-6 was detected using the ECL detection system.
Complex Formation between I␣I and TSG-6 -Complexes were produced in 50 mM Tris-HCl, 100 mM NaCl, pH 7.4, by incubating I␣I and TSG-6 at molar ratios ranging from 1:0.1 to 1:8 for 60 min at 37°C.
Complex Formation between HA, I␣I, and TSG-6 -Approximately 35 g of HA was mixed with 3.5 g of I␣I in 50 mM Tris-HCl, 100 mM NaCl, pH 7.4. Subsequently, 0.6 g of TSG-6 was added producing an I␣I: TSG-6 molar ratio of 1:1. The sample was incubated for 2 h at 37°C and then prepared for SDS-PAGE. Before SDS-PAGE the sample was filtered using a 0.2-m microspin filter (Lida). Hyaluronidase Digestion and NaOH Treatment of the I␣I⅐TSG-6 Complex and the HA⅐I␣I Complex-Hyaluronidase was dissolved in 20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.0. The I␣I⅐TSG-6 complex was made as described above using an I␣I/TSG-6 molar ratio of ϳ1:8. The I␣I⅐TSG-6 complex was digested using 0.06 unit of hyaluronidase/20 pmol of I␣I. The reaction was continued for 5 h at 37°C. NaOH dissociation of I␣I, I␣I⅐TSG-6 complexes, or HA⅐I␣I complex were performed as described before (5).
In-gel Digestion-Bands of interest were excised from Coomassie Blue-stained 7.5% SDS-PAGE gels, cut in small cubes, and washed in water. The gel pieces were then incubated in acetonitrile and rehydrated in 0.1 M ammonium bicarbonate. Finally, the gel pieces were swelled in 50 mM ammonium bicarbonate containing 25 g/ml trypsin before ammonium bicarbonate was added to cover the pieces. The samples were digested for ϳ16 h at 37°C. Following digestion, the tryptic peptides were extracted, filtered, and acidified prior to the mass spectrometry analysis.
Liquid Chromatography Tandem Mass Spectrometry-The LC-MS/MS analyses were performed using a Micromass Q-TOF Ultima Global mass spectrometer (Micromass/Waters) connected to a LC-Packings UltiMate nano LC System (LC-Packings). A nano-spray ion source was used to hold the packed PicoFrit TM columns (New Objective) and apply capillary voltage through a Valco union. The PicoFrit TM columns (75-m ID ϫ 10 cm) were packed with Zorbax SB C18, 3.5-m reverse phase column material (Agilent) using a high pressure column Loader (Proxeon). The column was developed at a flow rate of 200 nl/min and linear gradients from 0.02% heptafluorobutyric acid/0.5% acetic acid in water (Buffer A) to 0.02% heptafluorobutyric acid/0.5% acetic acid in 75% acetonitrile/24.5% water (Buffer B). After data acquisition, the individual MS/MS spectra acquired for each of the precursors within a single LC run were combined, smoothed, deisotoped, and centroided using the Micromass Masslynx data processing software and output as a single Mascot-searchable peak list. The peak list files were used to query the Swiss-Prot data base using the Mascot program (25).
Determination of Whether Both Forms of TSG-6 Are Capable of Forming a Covalent Complex with the HCs-I␣I⅐TSG-6 complexes were produced as described above and analyzed by reduced SDS-PAGE. The upper reservoir buffer composition was altered by adding only 0.05% SDS and by including 25 mg/liter Coomassie Brilliant Blue to facilitate staining of the proteins during electrophoresis (26). After the SDS-PAGE the proteins were electrophoretically transferred to a Biodyne B membrane (27). The section containing the bands, including HC1⅐bikunin, HC2⅐bikunin, HC1⅐TSG-6, and HC2⅐TSG-6 complexes, were excised and the membrane pieces were treated with NaOH (5). The proteins were extracted in sample buffer and analyzed by reduced SDS-PAGE followed by immunoblotting using anti-TSG-6 polyclonal antibodies.
Proteolytic Fragmentation of the HC2⅐TSG-6 Complex-Purified I␣I and TSG-6 was incubated as described above and separated by unreduced SDS-PAGE. The HC2⅐TSG-6 complex was electroeluted (28), and the recovered protein material was then digested with thermolysin. TSG-6 was more resistant to proteolysis than the HCs and conditions were established where the HCs were digested leaving TSG-6 intact. The reaction products were separated by a second SDS-PAGE analysis and electrophoretically transferred to an Immobilon-P membrane. After Coomassie Blue staining of the membrane, the band corresponding to TSG-6 was excised and analyzed by automated Edman degradation. NH 2 -terminal Sequence Analysis-Automated Edman degradation was performed in an Applied Biosystems 477A sequencer with on-line phenylthiohydantoin analysis using an Applied Biosystems 120A HPLC system operated according to the manufacturer's recommendations.

Incubation of I␣I and TSG-6 Produce Seven New Protein
Bands in SDS-PAGE-The incubation of purified I␣I and TSG-6 resulted in the appearance of seven protein bands when analyzed by reduced SDS-PAGE (Fig. 1). To aid in the description we used the following nomenclature: numbers refer to new bands that are covalently associated protein products of the reaction. These include HMW I␣I (bands 1 and 2), HC2⅐TSG-6 (band 3), and HC1⅐TSG-6 (band 4). Letters refer to bands that appear to be the result of dissociation of one or more I␣I components, including HC2⅐bikunin (band A), HC1⅐bikunin (band B), and free bikunin (band C).

Bikunin-containing Complexes and Free Bikunin (Bands A-C) Are Generated during the Incubation of I␣I with TSG-6 -
The bikunin-containing bands formed during the incubation of I␣I and TSG-6 were identified by non-reducing SDS-PAGE followed by TIC staining (Figs. 1 and 2). This technique enables the unambiguous identification of bikunin in contrast to Coomassie Blue staining (5). A fixed concentration of I␣I was incubated with increasing amounts of TSG-6. When equal amounts or a molar excess of TSG-6 was used (Fig. 2, lanes  7-9), a broad band about 30 -45 kDa in size showed trypsin inhibitory activity (Fig. 2, band C). This band represents free bikunin carrying the CS chain (4,8). Released bikunin does not migrate as a sharp band due to the heterogeneity of the attached CS chain. In addition to free bikunin two ϳ120-kDa bikunin-containing bands ( Figs. 1 and 2, bands A and B) were observed. The upper band (band A) was significantly more intense than the lower band (band B) (lanes 4 -6). The identity of the bikunin-containing complexes were established by a combination of (i) the apparent molecular mass in SDS-PAGE, (ii) immunoblotting using HC1, HC2, or bikunin-specific antibodies (data not shown), and (iii) the dissociation of both complexes by CS-degrading enzymes (Fig. 3, lanes 4 and 5). Based on these results we conclude that band A is composed of HC2⅐bikunin and band B of HC1⅐bikunin. These data show that free bikunin, HC1⅐bikunin, and HC2⅐bikunin are generated during the reaction between I␣I and TSG-6. We note that a significantly smaller amount of HC1⅐bikunin was consistently produced as compared with HC2⅐bikunin.
High Molecular Weight Bands (Bands 1 and 2) Are Generated during Incubation of I␣I and TSG-6 -Following the incubation of purified I␣I and TSG-6, two high molecular weight protein bands appear in both reduced and non-reduced SDS-PAGE ( Fig. 1-5, bands 1 and 2). TIC gel analysis revealed that both protein bands contain bikunin (Figs. 1 and 2). If I␣I was incubated with a molar excess of TSG-6 only very small amounts of these high molecular weight proteins were apparent (Fig. 2, lane 9, and unpublished data). We noticed that a smaller amount of the upper HMW I␣I band was produced compared with the lower HMW I␣I band. To determine the polypeptide composition, the two bands were excised, digested with trypsin, and subjected to LC-MS/MS. The analysis revealed the presence of bikunin, HC1, and HC2 in both bands (Table S1, Supplementary Material). No evidence for other proteins was apparent. We conclude that the high molecular weight proteins are made of the same components as I␣I (bikunin, HC1, and HC2) (1). The slower than expected migration in SDS-PAGE is most likely caused by a change in the bikunin/HC1/HC2 stoichiometry as compared with I␣I. The molecular weight of the faster migrating HMW I␣I band was estimated by reduced SDS-PAGE to be 71 kDa higher than I␣I (data not shown). An increase of 71 kDa is consistent with the After the incubation of I␣I with TSG-6 the sample was treated with NaOH prior to electrophoresis (lane 6). The bands are numbered and given letters according to Fig. 1. Both the high molecular weight proteins and the complexes between I␣I and TSG-6 are sensitive to mild NaOH treatment.
addition of one more HC as compared with I␣I. The slower migrating HMW I␣I band similarly displayed an increase in size as compared with the faster migrating HMW I␣I band. This suggests that HMW I␣I are formed in a process where additional HCs are cross-linked to the CS-chain. We designate these reaction products HMW I␣I.

Characterization of TSG-6-containing Protein Complexes (Bands 3 and 4) Generated during Incubation of I␣I with TSG-6 -
The I␣I⅐TSG-6 complexes are resistant to both hyaluronidase (Fig. 3, lane 5, bands 3 and 4) and chondroitinase ABC degradation (data not shown). These properties were exploited to separate the TSG-6-containing bands from the co-migrating bikunin-containing bands in SDS-PAGE (HC1⅐bikunin, band B; HC2⅐bikunin, band A). After the digestion, HC1⅐TSG-6 and HC2⅐TSG-6 remained at the same position in the gel, while HC1⅐bikunin and HC2⅐bikunin dissociated and migrated further into the gel (data not shown). The TSG-6-containing bands were subsequently subjected to LC-MS/MS (Fig. 3, lane 5,  bands 3 and 4). The analyses revealed that band 3 contained HC2 and TSG-6, whereas band 4 contains HC1 and TSG-6 (Table S1, Supplemental Material). We noted that significantly smaller amounts of HC2⅐TSG-6 as compared with HC1⅐TSG-6 were generated consistently during complex formation.
Stability of the HMW I␣I Cross-links-The nature of the HMW I␣I cross-links (bands 1 and 2) were investigated by analyzing their stability toward enzymatic or chemical dissociation protocols (Figs. 3 and 4). The complexes were dissociated by hyaluronidase (Fig. 3), chondroitinase ABC (data not shown), or by mild NaOH treatment (Fig. 4). This behavior is analogous to previous results obtained during the analysis of the PGP cross-link in I␣I and P␣I (5,8) suggesting that PGP cross-links are responsible for the cross-linking of HMW I␣I.
Stability of the HC(1 or 2)⅐TSG-6 Cross-links-The cross-link stabilizing the HC(1 or 2)⅐TSG-6 interaction was investigated as described above for the HMW I␣I proteins, including (i) stability toward hyaluronidase (Fig. 3), (ii) chondroitinase ABC (data not shown), and (iii) mild NaOH treatment (Fig. 4). Significantly, the HC(1 or 2)⅐TSG-6 cross-links were readily disrupted following NaOH treatment but was resistant to CSdegrading enzymes (Figs. 3 and 4, bands 3 and 4). Because NaOH dissociated the complex but CS-degrading enzymes did not, we conclude that an ester bond independent of the CS chain mediates the interaction. In addition the interaction between I␣I and TSG-6 produced HC1⅐bikunin and HC2⅐bikunin as described above. These are generated at the same time as the HC2⅐TSG-6 or HC1⅐TSG-6 complexes are formed (Fig. 1). Because HC1 is positioned closest to the nonreducing end of the CS (8) the formation of HC1⅐bikunin is impossible if cross-link formation involved cleaving the CS chain. Accordingly, the formation of HC2⅐TSG6 or HC1⅐TSG-6 does not involve cleavage of the CS chain.
The C-terminal Asp of HC2 Is Involved in the HC2⅐TSG-6 Cross-link-The I␣I⅐TSG-6 complex was prepared as described and separated by non-reducing SDS-PAGE. The HC⅐TSG-6 band was electroeluted and digested with thermolysin. TSG-6 was more resistant to proteolysis than the HCs, and conditions were established where TSG-6 remained intact following thermolysin digestion. The HCs were degraded, and the digest was separated by reduced SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane. N-terminal protein sequence analysis of the TSG-6 band revealed two sequences: the sequence LAQGSQVLESTPPPHVMRVEN(D) (Asp was not detected), corresponding to the C terminus of HC2, and WGFK-DGIFHNSIWLERAAGVYH, which matches the N-terminal sequence of TSG-6. All residues except Asp-702 of HC2 were detected during Edman degradation suggesting that the Cterminal Asp-702 is involved in the HC2⅐TSG-6 cross-link.
HA Inhibits the Formation of HMW⅐I␣I-The effect of HA on the formation of HMW⅐I␣I was investigated as described under "Experimental Procedures." When I␣I and TSG-6 were incubated in the presence of HA the formation of HMW⅐I␣I was abolished (Fig. 5, lane 3). In addition I␣I, HC1⅐bikunin, HC2⅐bikunin, HC1⅐TSG-6, and HC2⅐TSG-6 were not observed (Fig. 5, lane 3). The formation of a HA⅐HC complex is the most likely explanation for this observation (16,29). These complexes are large and will not migrate into the gel. However, the interaction between HA and the HCs are mediated by an ester (30), which is vulnerable to mild NaOH treatment. Significantly, we observed that mild NaOH treatment of complex between I␣I, TSG-6, and HA produced free HCs (Fig. 5, lane 4). The result shows that incubating I␣I and TSG-6 in the presence of HA generates HA⅐HC complexes and inhibits the formation of HMW I␣I.
Both the Glycosylated and the Non-glycosylated Forms of TSG-6 Are Capable of Forming a Covalent Complex with the HCs-It has been suggested that the two TSG-6 bands (Figs. 1, 3-5) are caused by differences in glycosylations (18). To determine if both TSG-6 glyco-forms were able to participate in complex formation I␣I⅐TSG-6 was separated by reducing SDS-PAGE and blotted to a Biodyne B membrane. The protein bands representing the HC1⅐bikunin, HC2⅐bikunin, HC1⅐TSG-6, and HC2⅐TSG-6 complexes were excised and treated with NaOH. The proteins were extracted from the membrane, and finally the sample was subjected to reduced SDS-PAGE and immunoblotting using anti-TSG-6. The result shows that both the glycosylated and the nonglycosylated forms of TSG-6 are able to form covalent interactions with the HCs (Fig. 6). The I␣I⅐TSG-6 complex was analyzed by reduced SDS-PAGE and transferred to a Biodyne B membrane. The bands representing the HC1⅐bikunin, HC2⅐bikunin, HC1⅐TSG-6 and HC2⅐TSG-6 complexes were excised and subjected to a mild NaOH treatment. The proteins were extracted and analyzed by reduced SDS-PAGE and immunoblotting using anti TSG-6 polyclonal antibodies (lane 1). TSG-6 was analyzed alone for comparison (lane 2). The glycosylated and the nonglycosylated forms of TSG-6 were both able to participate in the covalent complex formation.

DISCUSSION
When I␣I and TSG-6 interact apparent covalent interactions between TSG-6 and components of I␣I are formed (18,31,32). The mechanism of the reaction and the identity of the products are not clear (18,20). In the present study we have analyzed the polypeptide compositions of the reaction products, the chemical properties of the cross-links formed, and the reaction mechanism.
Characterization of the Reaction Products-Immunoblotting using TSG-6 antibody demonstrated that two TSG-6-containing complexes were formed during the incubation. In addition we show that HC1⅐bikunin, HC2⅐bikunin, free bikunin, and HMW I␣I were produced (Fig. 7). In contrast to a previous study (18) we specifically dissociated the HC1⅐bikunin and HC2⅐bikunin complexes before SDS-PAGE. The I␣I⅐TSG-6 complexes were resistant to both hyaluronidase and chondroitinase ABC, whereas HC1⅐bikunin and HC2⅐bikunin were readily dissociated. This prevented co-migration of HC(1 or 2)⅐bikunin and the two TSG-6 containing complexes. Subsequently, LC-MS/MS analyses of the two TSG-6-containing bands then revealed that they only were composed of HC1⅐TSG-6 or HC2⅐TSG-6 (Table S1, Supplementary Material). Bikunin is not involved in the TSG-6-containing complexes as suggested previously (18). The conflicting results reported previously (18,20) have led to the suggestion that different complexes between I␣I and TSG-6 exist in vivo (20). Although we are not able to explain the lack of HC1 or chondroitinase ABC sensitivity observed in a previous study (18), our data support that coincubation of I␣I and TSG-6 produce only HC1⅐TSG-6 and HC2⅐TSG-6 complexes.
In addition to the TSG-6-containing complexes the reaction produced other covalent protein complexes that we designate HMW I␣I. These bands did not contain TSG-6 and were shown to be composed of the same components as I␣I (Table S1, Supplementary Material, and Fig. 7). The bands behaved like I␣I in terms of trypsin inhibitory activity and dissociation by both chemical and enzymatic procedures. They are likely the result of HC transfer from one I␣I molecule to another during the reaction. Both the production of HC(1 or 2)⅐TSG-6 complexes and HMW I␣I thus consume HCs and the detection of HC1⅐bikunin, HC2⅐bikunin, and free bikunin suggests that I␣I served as a HC donor. In support of this we showed that more HC1⅐TSG-6 than HC2⅐TSG-6 was produced during the reaction (Fig. 3). Significantly, this was correlated with the formation of less HC1⅐bikunin than HC2⅐bikunin (Fig. 2). It is possible that the difference in the amount produced is caused by steric hindrance as HC2 is positioned closets to the reducing end of the CS (8). This could produce different environments for the two HCs and affect their ability to participate in the reaction.
In the present study we did not attempt to remove trace amounts of divalent cations from our protein preparations or buffer. Thus, we cannot out rule that the TSG-6⅐I␣I complex formation is a metal ion-dependent process.
The HC(1 or 2)⅐TSG-6 Cross-link-Several lines of evidence suggest that the cross-linking of I␣I and TSG-6 involves a transesterification where the PGP cross-link is cleaved, including (i) the cross-linking reaction dissociates I␣I producing free bikunin and HC(1 or 2)⅐bikunin (Fig. 2); (ii) HC⅐TSG-6 is not dissociated by CS-degrading enzymes suggesting that the cross-link does not contain [GlcUA-GalNAc] disaccharides (Fig.  3); (iii) the complexes are readily dissociated by mild NaOH treatment suggesting an ester bond (Fig. 4); and (iv) the HC2⅐TSG-6 cross-link was shown to be between the C-terminal Asp residue of HC2 and TSG-6. The most likely scenario is that during complex formation the ester bond between the ␣-carbon of the C-terminal Asp and the carbon-6 of an internal Nacetylgalactosamine is cleaved (Fig. 8A). A new ester bond is formed between the C-terminal Asp residues of the HCs and a TSG-6 residue containing a functional hydroxyl group, including Tyr, Ser, Thr, or a carbohydrate moiety (Fig. 8A). It has been suggested that the two TSG-6 bands observed following reduced SDS-PAGE, are glycosylated and non-glycosylated TSG-6 (18). The fact that both forms are involved in the covalent complex formation (Fig. 6) suggests that TSG-6 hydroxyl groups are not carbohydrate-derived.
The HMW I␣I Cross-link-The cross-links stabilizing HMW I␣I are different from the HC⅐TSG-6 cross-link. The HC⅐TSG-6 cross-link was resistant to CS-degrading enzymes, whereas HMW I␣I readily dissociated and the HMW I␣I cross-links are likely analogous to the PGP cross-link of I␣I (Fig. 3). Recently it has been demonstrated that TSG-6 is able to transfer HCs from I␣I to unsulfated chondroitin (29). We have previously shown that the bikunin CS is heterogeneously sulfated (8). The sulfate groups were mainly associated with disaccharides near the reducing end while the CS at the non-reducing end did not carry sulfate groups. It is possible that HMW I␣I is formed by a similar process where HCs are transferred from the CS of one I␣I molecule to the unsulfated CS region of another I␣I molecule (Fig. 8B). Consequently, I␣I molecules with more than two HCs are produced, accounting for the higher molecular weight (Fig. 7). HA is a better HC acceptor than chondroitin (29), and the fact that HA abolished the formation of HMW I␣I (Fig. 5), provides additional evidence for this hypothesis.
Mechanism of Complex Formation-The cross-linking reaction did not cleave the CS chain. If cleavage of the CS chain was part of the mechanism HC1⅐bikunin would not have been de- FIG. 7. Protein products formed during the incubation of I␣I with TSG-6. Co-incubation of I␣I and TSG-6 produces at least seven protein products apparent on SDS-PAGE. In the present work we have analyzed the composition of these products. This figure summarizes our findings. The proteins are numbered and given letters according to Fig. 1.  1 and 2, HMW I␣I; 3, HC2⅐TSG-6; 4, HC1⅐TSG-6; A, HC2⅐bikunin; B, HC1⅐bikunin; and C, bikunin. The structure of the HMW I␣I is a proposed structure based on our results. The exact composition and organization of the components in the HMW I␣I remain to be investigated. tected according to the position of the HCs on the CS (8). In addition CS-degrading enzymes were unable to dissociate the HC(1 or 2)⅐TSG-6 complex (Fig. 3) implying that [GlcUA-Gal-NAc] disaccharides were not involved in the cross-link. This is in contract to the HMW I␣I complex, which was readily cleaved by CS-degrading enzymes. Based on these findings the mechanism is likely to involve the formation of an intermediate that partitions to generate HC(1 or 2)⅐TSG-6 complexes or transfer HCs to CS via competing pathways. In favor of this mechanism two different covalent products are produced (HC⅐TSG-6 and HMW I␣I) and a progressively lower level of HMW I␣I was generated when a fixed concentration of I␣I was titrated with increasing amount of TSG-6 ( Fig. 2).
Taken together the data suggest that the cross-linking of I␣I and TSG-6 involves a transesterification where the PGP crosslink is cleaved. A new ester bond is formed between the Cterminal Asp ␣-carbonyl of the HCs and a TSG-6-derived hydroxyl donor most likely a Tyr, Ser, or Thr residue or possibly a glycan moiety. In a competing reaction HCs are transferred to the CS chain of an I␣I molecule by forming a new ester bond between C-terminal Asp ␣-carbonyl of HCs and a carbon-6 of an internal N-acetylgalactosamine of the CS producing a new PGP cross-link (Fig. 8).