Characterization of complexes formed between TSG-6 and inter-α-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (I alpha I), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC center dot HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human I alpha I and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 center dot HC1 and TSG-6 center dot HC2) act as intermediates in the formation of HC center dot HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact I alpha I is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 center dot HC1 and TSG-6 center dot HC2 complexes was accompanied by the production of bikunin center dot HC2 and bikunin center dot HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 center dot HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with I alpha I to form new TSG-6 center dot HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC center dot HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.