The Transfer of Heavy Chains from Bikunin Proteins to Hyaluronan Requires Both TSG-6 and HC2*

Tumor necrosis factor-stimulated gene-6 protein (TSG-6) is involved in the transfer of heavy chains (HCs) from inter-α-inhibitor (IαI), pre-α-inhibitor, and as shown here HC2·bikunin to hyaluronan through the formation of covalent HC·TSG-6 intermediates. In contrast to IαI and HC2·bikunin, pre-α-inhibitor does not form a covalent complex in vitro using purified proteins but needs the presence of another factor (Rugg, M. S., Willis, A. C., Mukhopadhyay, D., Hascall, V. C., Fries, E., Fülöp, C., Milner, C. M., and Day, A. J. (2005) J. Biol. Chem. 280, 25674–25686). In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including IαI, HC2·bikunin, and free HC2 promoted the formation of HC3·TSG-6 and subsequently HC3·hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.

Tumor necrosis factor-stimulated gene-6 protein (TSG-6) 2 is a multifunctional protein involved in inflammation and tissue remodeling (1). The 35-kDa protein is composed of a link and a CUB domain. The link domain mediates binding to glycosaminoglycans via two distinct binding sites (2,3). The CUB domain in TSG-6 is less studied, but it has been shown recently that the domain mediates interaction with fibronectin (4). The bikunin proteins inter-␣-inhibitor (I␣I), pre-␣-inhibitor (P␣I), and HC2⅐bikunin contain the serine proteinase inhibitor bikunin, which is a member of the I2 Kunitz-BPTI inhibitor family (MEROPS data base) (5,6). Bikunin carries a single chondroitin sulfate (CS) chain originating from Ser-10 (7) composed of both unsulfated chondroitin and chondroitin 4-sulfate (8,9). Furthermore the bikunin proteins contain different heavy chains (HCs) selected from a group of five proteins (10), but only three of the five homologous HCs have been identified in complex with bikunin. I␣I contains HC1 and HC2, P␣I contains HC3, and HC2⅐bikunin contains HC2 (11,12). The HCs are covalently attached to the CS chain originating from bikunin (7,11). This unique cross-link has been designated a protein-glycosaminoglycan-protein cross-link (7).
TSG-6 and I␣I HCs spontaneously form covalent complexes in vitro (13). The formation of these complexes involves a twostep process in which TSG-6 and the CS chain of bikunin initially interact in a divalent cation-independent way (14). Subsequently the HC⅐TSG-6 complexes are formed during a divalent cation-dependent transesterification reaction (14). These HC⅐TSG-6 complexes are intermediates in a process in which the HCs are transferred to hyaluronan (HA) (15).
Purified P␣I and TSG-6 do not form a covalent complex (15), although HC3⅐HA complexes exist in vivo (16). However, the reaction will take place in the presence of serum (15) suggesting that the formation of HC3⅐HA requires another component (15). In the present study we purified this component from human blood and identified it as HC2. Both HC1 and HC3 failed to promote the TSG-6 transfer activity, and the data show that both HC2 and TSG-6 are required for transfer activity to be expressed.

EXPERIMENTAL PROCEDURES
Materials-Magnesium chloride, HA sodium salt from human umbilical cord, and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Sigma-Aldrich. Chondroitinase ABC (EC 4.2.2.20) was either from Sigma-Aldrich or from Seikagaku. CNBr-activated Sepharose, ECL Western blotting reagents, and Mono Q 4.6/100 PE column were obtained from GE Healthcare. Mass spectrometry grade trypsin was from Promega. Stagetips (C 18 ) were from Proxeon Biosystems A/S. Antisera against TSG-6, HC1, HC3, and bikunin were produced as described before (11,17,18). With some modifications I␣I, P␣I, and HC2⅐bikunin were purified from human plasma also as described before (12). The plasma was obtained from Statens Serum Institut, Copenhagen, Denmark. CS substituted bikunin was purified from NaOH-dissociated I␣I as described recently (14). Human TSG-6 was expressed in insect cells and purified as described before (19). Protein concentrations were determined spectrophotometrically at 280 nm using theoretical extinction coefficients calculated by General Protein/Mass Analysis for Windows (GPMAW) software (Lighthouse Data).
Purification of HA Oligosaccharides-HA (10 mg) was digested with 0.5 units of chondroitinase ABC for 1 h at 37°C. The enzyme was inactivated by boiling and removed by reverse phase high pressure liquid chromatography (Aquapore RP-300) using water as the mobile phase. The flow-through was collected and applied to an anion exchange column (Mono Q 4.6/100 PE column, GE Healthcare) equilibrated in 20 mM Tris-HCl, pH 7.4 (buffer A). To separate the HA oligosaccharides the column was developed using a linear gradient from 0 to 0.4 M NaCl in buffer A. Relevant fractions were pooled, concentrated, and desalted by size exclusion chromatography (Superdex Peptide 10/300 GL (GE Healthcare) equilibrated in water) (22). The masses of the collected oligosaccharides were determined by MALDI time-of-flight mass spectrometry (Bruker Ultraflex, Bruker Daltonics, Bremen, Germany). The instrument was operated in linear and positive polarity mode using sinapinic acid (20 g/liter) as matrix. The concentration was determined by measuring the absorbance at 232 nm using 5500 cm Ϫ1 M Ϫ1 as the extinction coefficient (23).
Purification of Plasma Component Required for HC3⅐TSG-6 Complex Formation-Human plasma (3 ml) was diluted 3-fold in 20 mM Tris-HCl, pH 7.4 containing 20 mM EDTA. The sample was applied to a 5-ml HiTrap Q column (GE Healthcare) equilibrated in 20 mM Tris-HCl, 10 mM EDTA, pH 7.4 (buffer A) and operated at a flow rate of 2 ml/min. The column was developed using a 50-min 0 -0.5 M NaCl gradient in buffer A. The eluate was monitored at 280 nm, and five fractions of 20 ml were collected. The collected fractions were dialyzed against 20 mM Tris-HCl, 140 mM NaCl, pH 7.4 and assayed for the ability to transfer HC3. A fixed amount of protein (2.5 g) from each fraction was incubated with 0.3 g of TSG-6 and 0.3 g of P␣I in the presence of 1 mM MgCl 2 . Complex formation was monitored by reducing SDS-PAGE and immunoblotting using an anti-TSG-6 antibody. The fractions containing the HC3 transfer activity were pooled comprising a total volume 40 ml. The sample was concentrated (Centriprep YM-10, Amicon, Millipore) 20-fold, and 10% of the concentrate was subsequently applied to a size exclusion column (Superose 6, GE Healthcare) equilibrated in 20 mM Tris-HCl, 140 mM NaCl, pH 7.4. The column was eluted using a flow rate of 0.5 ml/min, and fractions of 1 ml were collected and assayed for HC3 transfer activity as described above. Active fractions were subjected to reducing SDS-PAGE and stained using Coomassie Blue before proteins were identified by peptide mass fingerprinting.
Formation of HC⅐TSG-6 Complexes-Bikunin proteins and TSG-6 were incubated in 50 mM Tris-HCl, 100 mM NaCl, pH 7.5 containing 1 mM MgCl 2 for 1 h at 37°C at a 1:1 molar ratio. The complex formation was visualized directly by reducing SDS-PAGE and immunoblotting using an anti-TSG-6 antibody. Alternatively the complex-containing sample was subsequently treated with chondroitinase ABC for 2 h at 37°C before the resulting products were visualized by reducing SDS-PAGE and immunoblotting using anti-HC1, anti-HC1 and -2, anti-HC3, and anti-TSG-6 antibodies.
TSG-6-mediated HC to HA Transfer Assay-The transfer experiment was performed as described above except that the proteins were incubated in a 25-fold molar excess of a 13-disaccharide long HA oligosaccharide (4940 Da). The transfer was observed by reducing SDS-PAGE. We chose one of the largest oligosaccharides that we were able to purify to homogeneity to obtain a clear band shift between free HCs and HC⅐HA.
Assay to Determine Whether Free HC2 Promotes HC3⅐TSG-6 Complex Formation-The role of free HC2 was analyzed by incubating purified HC2⅐bikunin (10 g) and 0.01 unit of chondroitinase ABC at 37°C overnight. A low concentration of enzyme was used to limit the chondroitinase ABC activity during subsequent complex formation. The digested sample was either used directly or heat-denatured, reduced, and S-carboxymethylated. P␣I, TSG-6, and free HC2 were incubated as described above. Complex formation was evaluated by reducing SDS-PAGE and immunoblotting using anti-TSG-6 antibodies. Similar to this approach we also treated HC2⅐bikunin with NaOH, as described before (7), to generate free HC2. Afterward we lowered the pH by the addition of 600 mM Tris-HCl, pH 7.6 and incubated the dissociated HC2⅐bikunin with P␣I and TSG-6. The final concentration of Tris in the sample and in the different controls for this experiment was 110 mM. Finally the complex formation was evaluated as described above.
Assay to Determine Whether Free HC1 Promotes HC3⅐TSG-6 Complex Formation-I␣I (0.5 mg) was treated with 1.5 units of chondroitinase ABC for 3 h at 37°C and applied to a Mono Q 4.6/100 PE column (GE Healthcare) equilibrated in 20 mM Tris-HCl, pH 7.4 (buffer A). Chondroitinase ABC did not bind to the column, and the dissociated I␣I components were eluted using a linear gradient from 0 to 0.8 M NaCl in buffer A and a flow rate of 1 ml/min. The fractions were collected manually, and a fixed volume of each collected peak was analyzed by reducing SDS-PAGE and visualized by silver staining or immunoblotting using an anti-HC1 antibody. Silver-stained protein bands were identified by peptide mass fingerprinting. The presence of chondroitinase ABC in the flow-through was confirmed by incubating with I␣I and analyzing the resulting reaction products by SDS-PAGE. An aliquot of each fraction was incubated with P␣I and TSG-6 as described above. HC3⅐TSG-6 complex formation was visualized by reducing SDS-PAGE and immunoblotting using an anti-TSG-6 antibody.
Assay to Determine Whether HC2 Is Required for the Transfer of HC3 from HC3⅐TSG-6 Complex to HA-Purified I␣I (3 mg) was immobilized on 2 ml of settled cyanogen bromide-activated Sepharose (GE Healthcare) according to the manufacturer's suggestions. The I␣I-Sepharose resin (50 l), 4 g of TSG-6, and 12 g of P␣I were then incubated for 2 h at 37°C in 200 l of 20 mM Tris-HCl, 137 mM NaCl, pH 7.4 containing 2 mM MgCl 2 . The I␣I-Sepharose was collected by gentle centrifugation, and the supernatant was removed. The supernatant (30 l) containing HC3⅐TSG-6 was incubated with either HA oligosaccharides (2 g) or with HA oligosaccharides (2 g) and HC2⅐bikunin (1.2 g) for 1 h at 37°C. Finally the samples were subjected to reducing SDS-PAGE and immunoblotting using anti-TSG-6 and anti-HC3 antibodies.
In-gel Digestion and Peptide Mass Fingerprinting Analysis-The in-gel digestion procedure was performed essentially as described before (13,24). Following digestion, the tryptic peptides were purified on a C 18 Stagetip (Proxeon Biosystems A/S), and eluted directly onto the MALDI target using 1 l of matrix (␣-cyano-4-hydroxycinnamic acid). The peptide mass fingerprints were recorded in a Micromass Q-Tof Ultima Global mass spectrometer (Waters). Mass spectra were acquired in positive ion mode (range, 800 -3000 m/z). Micromass Mass-Lynx data processing software was used to generate a single Mascot-searchable peak list to query the Swiss-Prot protein data base (25). The searches were performed using a peptide mass tolerance of 50 ppm or better and propionamide modification of cysteine residues and allowed one or no missed tryptic cleavage site. Only significant hits as defined by Mascot probability analysis were accepted.

RESULTS
Purification of the Plasma Component Required for HC3⅐ TSG-6 Complex Formation-Human plasma was separated by anion exchange chromatography (supplemental Fig. S1A), and the eluting fractions were assayed for HC3⅐TSG-6 cross-linking activity by immunoblotting. The HC3⅐TSG-6-promoting activity was present in the two last fractions from the anion exchange column. These fractions contain proteins that elute from 300 to 500 mM NaCl. The two fractions were pooled and subjected to size exclusion chromatography (supplemental Fig.  S1B), and the fractions were assayed for HC3⅐TSG-6 complex formation. Because the migration in SDS-PAGE of the three HCs is distinct (11), HC3⅐TSG-6, HC2⅐TSG-6, and HC1⅐TSG-6 could conveniently be distinguished. Because all three HC⅐TSG-6 complexes were present in the same sample, the HC3⅐TSG-6-promoting activity was apparently co-eluting with I␣I (Fig. 1A). The active fractions were further analyzed by reducing SDS-PAGE and peptide mass fingerprinting (Fig. 1, B and C, and supplemental Table S1). The results suggested that the protein corresponding to I␣I (band a) contained the promoting activity. Peptide mass fingerprinting analysis failed to detect bikunin in the band most likely due to ion suppression effects in the mass spectrometer of the bikunin-derived peptides. The presence of bikunin in the fraction (band a) was subsequently confirmed by reducing SDS-PAGE and immunoblotting using an antibikunin antibody verifying that the HC3⅐TSG-6-promoting band contains I␣I. I␣I and HC2⅐Bikunin Promote the Formation of HC3⅐TSG-6 Complex-To determine whether purified I␣I or purified HC2⅐bikunin were able to promote the formation of HC3⅐TSG-6 cross-links, P␣I, TSG-6, and I␣I or HC2⅐bikunin were incubated and analyzed by reducing SDS-PAGE and immunoblotting using an anti-TSG-6 antibody. Both I␣I and HC2⅐bikunin promoted the formation of HC3⅐TSG-6 complexes ( Fig. 2A).
To confirm the formation of HC3⅐TSG-6 by mass spectrometry I␣I, P␣I, and TSG-6 were incubated to form HC⅐TSG-6 complexes, and the reaction products were treated with chondroitinase ABC to dissociate co-migrating HC⅐bikunin proteins (13) and finally separated by reducing SDS-PAGE. The band of interest was excised and analyzed by peptide mass fingerprinting. These analyses confirmed the presence of HC3⅐TSG-6 (supplemental Table S2). Finally the chondroitinase ABCtreated sample and the chondroitinase ABC-treated I␣I and P␣I were analyzed by reducing SDS-PAGE and immunoblotting using antibodies directed toward the HCs and TSG-6 ( Fig.  2B). The samples were treated with chondroitinase ABC to simplify the interpretation of the results. The treatment dissociated the HC⅐bikunin complexes, but the HC1⅐HC2 complexes remain associated (Fig. 2B, lanes 4, 6, 10, and 12). In addition, the high molecular weight (HMW) I␣I forms are similarly not completely degraded (Fig. 2B, lanes 6 and 12). The immunoblot reveals that truncated versions of especially HC3 exist in our preparation (Fig. 2B, lanes 8 and 9). Most importantly the immunoblot shows that in addition to the anti-TSG-6 antibody (Fig. 2B, lane 3) the anti-HC3 antibody also detected HC3⅐TSG-6 (Fig. 2B, lane 9), confirming the presence of the complex. Taken together, the data demonstrate that  Table S1). Band "a" correlated with the observed level of transfer activity as it was found in fraction 15 and to a lesser extent fraction 16. The identification of HC1 and HC2 in band "a" and the migration of the band corresponded to I␣I. The result suggests that I␣I is responsible for the HC3⅐TSG-6 complex-promoting activity.
HC3⅐TSG-6 has been formed in vitro, and it is apparent that I␣I or HC2⅐bikunin are required for the HC3⅐TSG-6 complex formation to occur. These proteins most likely represent the serum component required for complex formation. I␣I and HC2⅐Bikunin Promote the TSG-6-mediated Transfer of HC3 from P␣I to HA-The transfer of HCs from bikunin proteins to HA is mediated by two sequential transesterifications, and the HC⅐TSG-6 complexes are intermediates in this process (15). Because I␣I and HC2⅐bikunin promoted HC3⅐TSG-6 complex formation we investigated whether HC3 was transferred to HA. P␣I, TSG-6, and HA oligosaccharides were incubated in the presence of I␣I and subjected to reducing SDS-PAGE. When this sample (Fig. 3A, lane 7) is compared with the incubation of I␣I, TSG-6, and HA it is apparent that a new band appears (Fig. 3A, lane 7, band a). The interactions between HA and HCs are mediated by an alkaline-sensitive ester, and the migration of the new band was sensitive to mild NaOH treatment (Fig. 3A, lane 8) supporting that the band represents HA⅐HC3. Indeed this was confirmed by peptide mass fingerprinting ( Fig. 3B and supplemental Table S3). Furthermore we found that HC2⅐bikunin similarly to I␣I promoted the TSG-6-mediated transfer of HC3 from P␣I to HA (supplemental Table S4). Taken together these experiments demonstrate that HC2⅐bikunin and I␣I promote the TSG-6-mediated transfer of HC3 from P␣I to HA.
TSG-6 Generates HMW P␣I-HMW proteins, containing bikunin and more than two HCs, are produced if bikunin proteins are incubated with TSG-6 ( Fig. 3A, lanes 4 and 5) (13). However, when an excess of HA is added, HA is used as HC acceptor rather than the bikunin CS chain, and the formation of HMW proteins is abolished (Fig. 3A, lanes 6 and 7). The HMW proteins previously have been designated HMW I␣I (13), but when/if I␣I, P␣I, and TSG-6 are incubated together (Fig. 3A, lane 5), the term HMW bikunin proteins is more appropriate.
This became apparent because immunoblotting, using an anti-HC3 antibody, revealed that HC3 was present both in the protein band corresponding to bikunin containing two HCs and in the protein band containing three HCs (data not shown) demonstrating that the TSG-6-mediated transfer of HCs also generates HMW P␣I. The level of HMW P␣I was, as expected, significantly reduced when I␣I, P␣I, and TSG-6 were incubated in the presence of HA. TSG-6 Transfers HC2 from Purified HC2⅐Bikunin to HA-We have shown that HC2⅐bikunin and I␣I promote the TSG-6-mediated transfer activity. These analyses indicated that although HC2⅐bikunin and P␣I are structurally similar, containing bikunin and one HC, they are functionally different. Therefore we tested whether purified HC2⅐bikunin inter-   1, 4, 7, and 10) and P␣I (lanes 2, 5, 8, and 11) were digested with chondroitinase ABC. Furthermore TSG-6, P␣I, and I␣I were incubated and subsequently treated with chondroitinase ABC to dissociate the protein-glycosaminoglycan-protein cross-links (lanes 3, 6, 9, and 12). The resulting reaction products were analyzed by SDS-PAGE and immunoblotting using primary antibodies (Ab) as indicated. The positions of the HC⅐TSG-6 complexes are shown by encircled bands. The results underline that P␣I and TSG-6 covalent complex formation occurs in the presence of I␣I. In addition the results demonstrate that the cross-link remains intact following chondroitinase ABC treatment.

B A
acted covalently with TSG-6. The bikunin proteins HC2⅐bikunin, P␣I, and I␣I were incubated with TSG-6, and the complex formation was evaluated by reducing SDS-PAGE and immunoblotting using an anti-TSG-6 antibody (Fig. 4A). The analysis revealed that HC2 derived from purified HC2⅐bikunin reacted covalently with TSG-6. This was also confirmed by peptide mass fingerprinting of the complex from a Coomassie Blue-stained gel (Fig. 4, B and C, and supplemental Table  S5). Like the complexes originating from I␣I, this complex was dissociated by mild NaOH treatment and required divalent cations during the formation (data not shown). The result demonstrated that covalent complex formation did not depend on the presence of two HCs attached to the CS chain for complex formation with TSG-6 to proceed. Furthermore we tested whether HC2 from purified HC2⅐bikunin was transferred to HA. TSG-6, HA oligosaccharides, and HC2⅐bikunin or P␣I were incubated. The samples were analyzed by reducing SDS-PAGE, and protein bands of interest were identified by peptide mass fingerprinting (Fig. 4, B and C, and supplemental Table S5). The results revealed that HC2 derived from HC2⅐bikunin was transferred to HA (Fig. 4B, lane 7, band b) and that the HC2⅐HA crosslink was dissociated by mild NaOH treatment (Fig. 4B, lane 9). In addition, HC2 derived from HC2⅐bikunin was also transferred to the CS originating from bikunin in the absence of HA, generating HMW HC2⅐bikunin proteins (Fig. 4B, lane 5). In contrast and as expected, no transfer of HCs to either CS or HA was observed when P␣I was incubated with TSG-6 Ϯ HA (Fig. 4B, lanes 4 and  6). These analyses demonstrate that P␣I and HC2⅐bikunin are functionally different.
Free HC2 Alone Promotes the Formation of HC3⅐TSG-6-In simple terms I␣I and HC2⅐bikunin are composed of bikunin, CS, and HCs. Bikunin is most likely not responsible for the HC3⅐TSG-6-promoting effect of I␣I and HC2⅐bikunin because P␣I contains bikunin. Indeed purified bikunin (containing the CS chain) was unable to promote the HC3⅐TSG-6 complex formation (data not shown). HC2 is the only other component shared by I␣I and HC2⅐bikunin. To investigate the role of HC2 alone, HC2⅐bikunin was dissociated by chondroitinase ABC treatment. Free HC2 did not form a covalent complex with TSG-6 (Fig. 5, lane 4) (14), but significantly, free HC2 promoted Subsequently the samples were analyzed by immunoblotting using anti-TSG-6 antibody. This analysis demonstrates that HC2⅐bikunin and I␣I interact covalently with TSG-6. B, TSG-6 was incubated with bikunin proteins and HA as indicated above the gel. The samples were separated by reducing SDS-PAGE and stained using Coomassie Blue. The HC2⅐bikunin preparation contained small amounts of intact and truncated free HC2 as indicated by the asterisk (*). C, the labeled bands were subjected to peptide mass fingerprinting (supplemental Table S5). Taken together, the mass spectrometry analysis, the migration of the proteins, and the sensitivity to NaOH treatment strongly indicate that band b corresponds to HC2⅐HA. The result shows that, in contrast to HC3 from P␣I, TSG-6 does transfer HC2 from HC2⅐bikunin to HA.  Table S6). D, finally the starting material (SM) before separation on the Mono Q column and the collected peak fractions (P1-P4) were analyzed by reducing SDS-PAGE and immunoblotting using anti-HC1 as primary antibody. Taken together, the analyses reveal that free intact and truncated HC1 was purified (P1). HC2 co-eluted with bikunin and the HC1⅐HC2 complex (P2-P4) preventing the purification of HC2 using this method. mAU, milliabsorbance units.
by immunoblotting using an anti-HC3 antibody (Fig. 8B, lane 5). Small amounts of HC3⅐HA were observed before the addition of HC2⅐bikunin (Fig. 8B, lane 4). This is likely to be explained by trace amounts of HC2, HC2⅐bikunin, or I␣I released from the Sepharose. The formation of HC3⅐TSG-6 was not observed on the anti-HC3 immunoblot because it co-migrates with P␣I and is present in a much smaller amount than P␣I. The anti-HC3 immunoblot clearly demonstrated the formation of the previously mentioned HMW P␣I (Fig. 8B, lanes [3][4][5]. These data suggest that HC2 is required for both the first and the second transesterification.

DISCUSSION
The present study confirms that TSG-6 alone is unable to transfer HC3 from P␣I to HA in vitro (15). This observation has been puzzling in view of the fact that HC3⅐HA has been observed in vivo (16), and studies with TSG-6 knock-out mice show that TSG-6 transfers HC1 and HC2 from I␣I and HC3 from P␣I to HA in vivo (26). Apparently another component is present in vivo that is required for the completion of the HC3 transfer (15). We show in this study that this component is HC2 and that TSG-6 in concert with HC2 is responsible for the TSG-6-mediated transfer of HC3 to HA.
The HCs exist in five variations (10,27). These all contain a vault protein inter-␣-trypsin domain and a von Willebrand type-A domain (10). Despite the sequence identity between the HCs, the present study demonstrates that significant functional differences exist.
Free HCs are not present in vivo, and although free HC2 promotes HC3⅐TSG-6 complex formation, the physiologically relevant proteins are HC2⅐bikunin and I␣I. Therefore, the colocalization of bikunin proteins, TSG-6, and HA is likely to ensure transfer of all HCs to HA in vivo. The primary means of regulation is probably at the level of expression and the localization of the reactants. Bikunin proteins are constitutively expressed and present in blood (6) in contrast to the expression of both TSG-6 and HA (1,28). The interaction between the bikunin proteins and TSG-6/HA is established when the bikunin proteins access the extracellular space, for instance during ovulation (29). If bikunin proteins and TSG-6 co-localize in the absence of HA or unsulfated chondroitin, which also acts as an HC acceptor (30), TSG-6 is likely to transfer the HCs to the CS chain of bikunin generating HMW I␣I and HMW P␣I.
HC2⅐bikunin is a consequence of the I␣I and TSG-6 interaction forming HC⅐TSG-6 complexes (13,15). In the present study we show that HC2⅐bikunin and TSG-6 generate a covalent complex. This demonstrates that the by-product of the first TSG-6, P␣I, and the peak fractions (P1-P4) derived from the anion exchange purification of HC1 (see Fig. 6) were incubated and analyzed by reducing SDS-PAGE. Various control samples were included as indicated. Bands of interest were visualized by immunoblotting using an anti-TSG-6 antibody. The immunoblot confirmed that the HC2-containing peaks (P2-P4) promote HC3⅐TSG-6 complex formation. In contrast, peak 1, which contains free HC1, did not promote the formation of HC3⅐TSG-6. . The experiment shows that immobilized I␣I promotes HC3⅐TSG-6 complex formation. Furthermore the result demonstrates that HC2 is required for the subsequent transfer of HC3 from HC3⅐TSG-6 to HA.
reaction could be used for subsequent HC⅐TSG-6 complex formation thereby releasing free bikunin. Previously it has been shown that free bikunin is generated during the I␣I/TSG-6 interaction as a result of HC⅐bikunin breakdown (15,31). Here we show that this alternative pathway also generates free bikunin as TSG-6 strips the CS chain of the HCs. This may impact the regulatory role of bikunin within the extracellular protease network (31,32).
In summary, we report that HC2⅐bikunin forms a covalent complex with TSG-6 and that HC2 is the additional factor required for the HC3⅐TSG-6 complex formation. Our studies show that HC2 and TSG-6 in collaboration promote the transfer of HCs from bikunin proteins to HA.