Evidence for a two-step mechanism involved in the formation of covalent HC x TSG-6 complexes

Biochemistry. 2006 Jun 20;45(24):7661-8. doi: 10.1021/bi060106s.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alpha-Globulins / chemistry
  • Alpha-Globulins / metabolism*
  • Binding Sites
  • Calcium / metabolism
  • Cations, Divalent / metabolism
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / metabolism*
  • Chelating Agents / pharmacology
  • Chondroitin Sulfates / metabolism
  • Edetic Acid / pharmacology
  • Esterification
  • Esters / metabolism
  • Kinetics
  • Magnesium / metabolism
  • Manganese / metabolism
  • Membrane Glycoproteins / metabolism
  • Molecular Weight
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Surface Plasmon Resonance
  • Trypsin Inhibitor, Kunitz Soybean / metabolism


  • Alpha-Globulins
  • Cations, Divalent
  • Cell Adhesion Molecules
  • Chelating Agents
  • Esters
  • Membrane Glycoproteins
  • SPINT2 protein, human
  • Manganese
  • Chondroitin Sulfates
  • Trypsin Inhibitor, Kunitz Soybean
  • Edetic Acid
  • Magnesium
  • Calcium