Characterization of Complexes Formed between TSG-6 andInter-α-inhibitor That Act as Intermediates in the Covalent Transfer ofHeavy Chains ontoHyaluronan

Abstract

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 (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.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 IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x 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 IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

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

“…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).…”

“…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.…”

“…In the first reaction, the ester bond, between the C termini of the HCs and the CS chain in I␣I is cleaved, and a new transient ester bond is formed between the C termini of the HCs and Ser-28 (numbered according to the preprotein) in TSG-6 (15,17). In the second transesterification, the transient ester bond is cleaved and reformed between the C terminus of the HCs and HA (14). HC2 is required during both transesterification reactions, but the molecular mechanism is currently unknown (16).…”

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation

“…The transfer of ITIHs onto HA requires tumour necrosis factor a-induced protein 6 (TNFAIP6), also known as TNF-stimulated gene 6 (Jessen and Odum, 2003). TNFAIP6 forms a stable complex (Rugg et al, 2005) with ITIH and HA during the transesterification reaction (Sanggaard et al, 2005). This formation of ITIH-HA complexes is thought to play an important role in the stabilization of HA-rich extracellular matrices, with TNFAIP6 acting as an essential cofactor and catalyst (Rugg et al, 2005).…”

“…TNFAIP6 forms a stable complex (Rugg et al, 2005) with ITIH and HA during the transesterification reaction (Sanggaard et al, 2005). This formation of ITIH-HA complexes is thought to play an important role in the stabilization of HA-rich extracellular matrices, with TNFAIP6 acting as an essential cofactor and catalyst (Rugg et al, 2005). To date, five distinct ITIHs have been identified, encoded by five genes located on two different chromosomes (DiarraMehrpour et al, 1989;Himmelfarb et al, 2004).…”