Evidence for a Two-Step Mechanism Involved in the Formation of Covalent HC·TSG-6 Complexes

Abstract

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.

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

“…1, lanes 1 and 3). Previously, it has been shown that the HC transfer reaction is HC2-and divalent cation-dependent (14,16,28). Therefore, samples without HC2 or without divalent cations were used as negative controls, and as expected, no HC transfer was observed in these samples (Fig.…”

“…HMW I␣I was also degraded by chondroitinase ABC, indicating that the HCs similarly had been rearranged. Moreover, inhibition of the TSG-6/HC2 transfer activity (14,28) prevented chondroitinase ABC from cleaving and releasing the two HCs, supporting that the authentic position was maintained (Fig. 3, lane 4).…”

“…Polyclonal rabbit anti-sera against bikunin and against HC1 and HC2 were produced as described previously (6). Free bikunin containing the CS chain was purified from NaOH dissociated I␣I as described before (28). HA oligosaccharides of distinct sizes were purified from a limited chondroitinase ABC digest of polymers of HA, as described recently (16).…”

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

“…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).…”

“…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).…”