title: Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan.
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.
authors.affiliation: Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Marilyn S
authors.initials: MS
authors.lastname: Rugg
authors.forename: Antony C
authors.initials: AC
authors.lastname: Willis
authors.forename: Durba
authors.initials: D
authors.lastname: Mukhopadhyay
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Hascall
authors.forename: Erik
authors.initials: E
authors.lastname: Fries
authors.forename: Csaba
authors.initials: C
authors.lastname: Fülöp
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Milner
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
mesh_terms: D000510:Alpha-Globulins / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002810:Chondroitinases and Chondroitin Lyases / Q000494:pharmacology; D004330:Drosophila; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012972:Sodium Hydroxide / Q000494:pharmacology
chemical_list: D000510:Alpha-Globulins; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; C072273:TNFAIP6 protein, human; C513057:Tnfaip6 protein, mouse; C011877:inter-alpha-inhibitor; D008345:Manganese; D012972:Sodium Hydroxide; D006820:Hyaluronic Acid; D002810:Chondroitinases and Chondroitin Lyases; D008274:Magnesium