IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2.

In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including I␣I, HC2⅐bikunin, and free HC2 promoted the formation of HC3⅐TSG-6 and subsequently HC3⅐hyaluronan 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.

Studies of the interaction between Bikunin proteins, tumor necrosis factor-stimulated gene-6 protein (TSG-6), and glycosaminoglycans have revealed a unique catalytic activity where TSG-6/heavy chain 2 transfer heavy chain subunits between glycosaminoglycan chains. The activity is mediated by TSG-6/ heavy chain 2 and involves a transient SDS stable interaction between TSG-6 and the heavy chain to be transferred. The focus of this study was to characterize the molecular structure of this cross-link to gain further insight into the catalytic mechanism. The result showed that the C-terminal Asp residue of the heavy chains forms an ester bond to Ser 28 ␤-carbon of TSG-6 suggesting that this residue plays a role during catalysis.