title: Evidence for a two-step mechanism involved in the formation of covalent HC x 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.
authors.affiliation: Center for Insoluble Protein Structure (inSPIN), Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Kristian W
authors.initials: KW
authors.lastname: Sanggaard
authors.forename: Carsten S
authors.initials: CS
authors.lastname: Sonne-Schmidt
authors.forename: Christian
authors.initials: C
authors.lastname: Jacobsen
authors.forename: Ida B
authors.initials: IB
authors.lastname: Thøgersen
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Valnickova
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Wisniewski
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
mesh_terms: D000510:Alpha-Globulins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002809:Chondroitin Sulfates / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004951:Esterification; D004952:Esters / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020349:Surface Plasmon Resonance; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism
chemical_list: D000510:Alpha-Globulins; D002413:Cations, Divalent; D015815:Cell Adhesion Molecules; D002614:Chelating Agents; D004952:Esters; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; D008345:Manganese; D002809:Chondroitin Sulfates; D014360:Trypsin Inhibitor, Kunitz Soybean; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium