Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

J Biol Chem. 2015 Nov 27;290(48):28708-23. doi: 10.1074/jbc.M115.669838. Epub 2015 Oct 14.


The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Keywords: CUB module structure; TSG-6; cumulus-oocyte complex expansion; heavy chain-hyaluronan complex formation; hyaluronan; inter-α-inhibitor; protein structure; protein-protein interaction; reproduction; site-directed mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Adhesion Molecules* / chemistry
  • Cell Adhesion Molecules* / genetics
  • Cell Adhesion Molecules* / metabolism
  • Cumulus Cells / metabolism*
  • Extracellular Matrix* / chemistry
  • Extracellular Matrix* / genetics
  • Extracellular Matrix* / metabolism
  • Humans
  • Hyaluronic Acid* / chemistry
  • Hyaluronic Acid* / metabolism
  • Mice
  • Oocytes / metabolism*


  • Cell Adhesion Molecules
  • TNFAIP6 protein, human
  • Tnfaip6 protein, mouse
  • Hyaluronic Acid

Associated data

  • PDB/1SZB