Characterization of Complexes Formed between TSG-6 andInter-α-inhibitor That Act as Intermediates in the Covalent Transfer ofHeavy Chains ontoHyaluronan
The high molecular mass glycosaminoglycan hyaluronan (HA) can becomemodified by the covalent attachment of heavy chains (HCs) derived from theserum protein inter-α-inhibitor (IαI), which is composed of threesubunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfatemoiety. The formation of HC·HA is likely to play an important role inthe stabilization of HA-rich extracellular matrices in the context ofinflammatory disease (e.g. arthritis) and ovulation. Here, we havecharacterized the complexes formed in vitro between purified humanIαI and recombinant human TSG-6 (an inflammation-associated proteinimplicated previously in this process) and show that these complexes(i.e. TSG-6·HC1 and TSG-6·HC2) act as intermediates inthe formation of HC·HA. This is likely to involve twotransesterification reactions in which an ester bond linking an HC tochondroitin sulfate in intact IαI is transferred first onto TSG-6 andthen onto HA. The formation of TSG-6·HC1 and TSG-6·HC2 complexeswas accompanied by the production of bikunin·HC2 and bikunin·HC1by-products, respectively, which were observed to break down, releasing freebikunin and HCs. Both TSG-6·HC formation and the subsequent HC transferare metal ion-dependent processes; these reactions have a requirement foreither Mg2+ or Mn2+ and are inhibited byCo2+. TSG-6, which is released upon the transfer of HCs from TSG-6onto HA, was shown to combine with IαI to form new TSG-6·HCcomplexes and thus be recycled. The finding that TSG-6 acts as cofactor andcatalyst in the production of HC·HA complexes has importantimplications for our understanding of inflammatory and inflammation-likeprocesses. The high molecular mass glycosaminoglycan hyaluronan (HA) can becomemodified by the covalent attachment of heavy chains (HCs) derived from theserum protein inter-α-inhibitor (IαI), which is composed of threesubunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfatemoiety. The formation of HC·HA is likely to play an important role inthe stabilization of HA-rich extracellular matrices in the context ofinflammatory disease (e.g. arthritis) and ovulation. Here, we havecharacterized the complexes formed in vitro between purified humanIαI and recombinant human TSG-6 (an inflammation-associated proteinimplicated previously in this process) and show that these complexes(i.e. TSG-6·HC1 and TSG-6·HC2) act as intermediates inthe formation of HC·HA. This is likely to involve twotransesterification reactions in which an ester bond linking an HC tochondroitin sulfate in intact IαI is transferred first onto TSG-6 andthen onto HA. The formation of TSG-6·HC1 and TSG-6·HC2 complexeswas accompanied by the production of bikunin·HC2 and bikunin·HC1by-products, respectively, which were observed to break down, releasing freebikunin and HCs. Both TSG-6·HC formation and the subsequent HC transferare metal ion-dependent processes; these reactions have a requirement foreither Mg2+ or Mn2+ and are inhibited byCo2+. TSG-6, which is released upon the transfer of HCs from TSG-6onto HA, was shown to combine with IαI to form new TSG-6·HCcomplexes and thus be recycled. The finding that TSG-6 acts as cofactor andcatalyst in the production of HC·HA complexes has importantimplications for our understanding of inflammatory and inflammation-likeprocesses. Hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; HC, heavy chain; IαI,inter-α-inhibitor; COC, cumulus-oocyte complex; CS, chondroitin sulfate;PαI, pre-α-inhibitor; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES,4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonicacid; p-HA, polymeric hyaluronan; HA14, hyaluronan 14-mer. isa high molecular mass polysaccharide that plays an important role inextracellular matrix structure and is central to a wide variety ofphysiological and pathological processes such as ovulation and inflammatorydisease (1Tammi M.I. Day A.J. Turley E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar,2Toole B.P. Nat. Rev.Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1697) Google Scholar). This glycosaminoglycan,which is composed solely of a repeating disaccharide of glucuronic acid andN-acetylglucosamine, can become covalently modified by the attachmentof heavy chains (HCs) from the serum proteoglycans of theinter-α-inhibitor (IαI) family; this modification is likely toalter both its matrix and solution properties(3Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Yingsung W. Zhuo L. Mörgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H.I. Kimata K. J. Biol. Chem. 2003; 278: 32710-32718Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 5Zhuo L. Hascall V.C. Kimata K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google the formation of HC·HA as the L. Hascall V.C. Kimata K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google is for of a HA-rich matrix that is the to L. Yoneda M. M. W. N. Y. K. Kimata K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google Scholar, D. Day A.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google matrix of the cumulus-oocyte is in M. Day A.J. L. 2004; PubMed Scopus Google Scholar). HC·HA complexes formed inflammatory in J. D. Google D. Yoneda M. S. Y. Ishiguro N. Iwata H. Kimata K. Google this to become W. Zhuo L. Mörgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H.I. Kimata K. J. Biol. Chem. 2003; 278: 32710-32718Abstract Full Text Full Text PDF PubMed Scopus (94) Google D. J. PubMed Scopus Google J. PubMed Scopus Google which is likely to and and by N. PubMed Scopus Google Scholar). IαI is an that protein and the bikunin) together via sulfate L. Hascall V.C. Kimata K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The is of via a glycosaminoglycan the HCs are linked ester between acid and the of in the W. M. M. J. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, H. PubMed Scopus Google HCs to be transferred onto by a become linked via an ester bond from to the of in M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (151) Google HC ester bond to in intact IαI protein is for this the the HCs are to IαI the which has a heavy to S. J. Full Text PDF PubMed Google form HC·HA in a L. Yoneda M. M. W. N. Y. K. Kimata K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google H. S. S. Y. N. N. K. H. H. J. PubMed Scopus Google Scholar). of purified in vitro to the formation L. Yoneda M. Kimata K. J. Biol. Chem. Full Text PDF PubMed Google are L. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has TSG-6 protein of as J. J. Biol. PubMed Scopus Google TSG-6 is an inflammation-associated protein of and J. PubMed Scopus Google Scholar, Day A.J. 2003; PubMed Scopus Google Scholar, J. 2004; PubMed Scopus Google to have an role in the transfer of HCs from S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google L. 2003; PubMed Scopus Google Day A.J. 2003; PubMed Scopus Google Scholar). S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google are to form the HA-rich extracellular matrix that is a that with the complexes in the of these the TSG-6, as a recombinant protein or as a of this in to and was from the that TSG-6 is for the transfer of onto S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google Scholar). The of TSG-6 is in the Day A.J. 2003; PubMed Scopus Google Scholar, Y. Hascall V.C. PubMed Scopus Google Scholar, S. S. S. PubMed Google Scholar, Day A.J. Biol. PubMed Scopus Google Scholar, D. Hascall V.C. Day A.J. PubMed Scopus Google Scholar, S. J. 2002; PubMed Scopus Google Scholar, Day A.J. 2003; PubMed Scopus Google the protein has shown to with and IαI in matrix Day A.J. Biol. PubMed Scopus Google D. Hascall V.C. Day A.J. PubMed Scopus Google Scholar). that TSG-6 is as a protein and as of that is with both Day A.J. Biol. PubMed Scopus Google D. Hascall V.C. Day A.J. PubMed Scopus Google Scholar). of by mass that and the of molecular TSG-6 is and HC is is likely to a of TSG-6·HC2 D. Hascall V.C. Day A.J. PubMed Scopus Google Scholar). complexes are to that the of IαI is in are by with the of ester the TSG-6·HCcomplexes act as intermediates in the formation of as S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google Scholar). to the this can be formed human recombinant TSG-6 and IαI are together in Day A.J. 2003; PubMed Scopus Google J. PubMed Scopus Google J. Day A.J. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google complexes of this have in of M. S. L. J. J. Google Scholar). the the formed in vitro from human was to TSG-6, and together via J. PubMed Scopus Google that a of TSG-6 an HC the with that formed ovulation in the this we have the complexes formed in purified human IαI and recombinant human TSG-6 and TSG-6·HC2 and have shown that act in the formation of HC·HA. This is accompanied by of and bikunin·HC2 to and HCs. Both TSG-6·HC and subsequent HC transfer are metal ion-dependent requirement for Mg2+ or TSG-6, which upon the transfer of HCs from TSG-6 onto HA, was shown to IαI to new TSG-6·HC complexes and thus acts as for the formation of HC·HA. and was in purified to as J. Day A.J. J. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and the was by acid IαI was human serum H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google and was as M. M. J. J. Biol. Chem. 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K. 2003; PubMed Scopus Google the of inhibited HC·HA and for IαI and were that transfer of HCs onto from both IαI and The of to the formation of HC·HA complexes with of IαI and the of a transfer for IαI was the of were This that the formation of HC·HA complexes from are this in which was with recombinant human TSG-6 and a of in our in vitro of complexes or the transfer of onto in this TSG-6 is formation of and that (e.g. is of TSG-6·HC for shown in in the reactions in which to be TSG-6·HC with transfer was the of was in in which high of (e.g. of were with TSG-6 and IαI of TSG-6·HC complexes the complexes were in that the TSG-6·HC complexes are upon the are likely to act as intermediates in of HCs onto HA. this and to of the TSG-6·HC these were formed and for a of the of and an of shown in in the of HA, was in the the in in was This that the TSG-6·HC complexes are a of in and this is likely to be the as in reactions of TSG-6 and IαI the of the TSG-6·HC that this from the of IαI and this which was in the was to and HC2 an The molecular mass of this and the that was formed the and of that is likely to is and HC2 both the was was to TSG-6·HC for a the was in the of the with this the both and HC2 and which is with of and were in the the likely to an with both The was shown to both HCs and HC2 This was formed in the and is likely to this a molecular mass is the of the likely in the of a HC·HA with was a in the of the in in which the formation of the TSG-6 was released upon the transfer onto HA. 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Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). with that or HC2 or to TSG-6·HC or HC·HA the or of The of complexes and TSG-6·HC2) from human and to are likely to be the as previously in D. Hascall V.C. Day A.J. PubMed Scopus Google Scholar). the of matrix in both and the of the HA-rich matrix via the HC·HA S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google D. Day A.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Day A.J. 2003; PubMed Scopus Google M. D. and M. Scholar). this the TSG-6 used in this can the of S. D. Day A.J. Hascall V.C. K. 2003; PubMed Scopus Google Scholar). that HC·HA is formed in the this be by Biol. 2004; PubMed Scopus Google is a to the of TSG-6·HC and HC·HA formation likely that these complexes form TSG-6, and TSG-6·HC can a wide of and acid that are with complexes of which are likely to and HC·HA have in of inflammatory processes (e.g. W. Zhuo L. Mörgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H.I. Kimata K. J. Biol. Chem. 2003; 278: 32710-32718Abstract Full Text Full Text PDF PubMed Scopus (94) Google J. D. Google D. Yoneda M. S. Y. Ishiguro N. Iwata H. Kimata K. Google J. PubMed Scopus Google M. S. L. J. J. Google and (e.g. ovulation in M. D. and M. and M. Day A.J. J. 2002; Full Text Full Text PDF PubMed Scopus Google this Day A.J. 2003; PubMed Scopus Google and PubMed Scopus Google in to inflammatory and IαI can from serum to J. D. Google J. PubMed Scopus Google or such as in the L. J. Biol. Chem. Full Text PDF PubMed Google Hascall V.C. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of complexes the an is this has that the of is in the formation of the L. PubMed Google Scholar). is a high of (e.g. that TSG-6·HC formed be and be an of in from is a of to the of M. S. L. J. J. Google Scholar). the TSG-6 acts as a in the transfer of HCs onto is likely that of TSG-6 to a of HC·HA which be of to to (e.g. is to the to the of and pathological and to the of for for with