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

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  • David C Briggs, University of Manchester, United Kingdom
  • Holly L Birchenough, University of Manchester, United Kingdom
  • Tariq Ali, University of Manchester, United Kingdom
  • Marilyn S Rugg, University of Oxford, United Kingdom., United Kingdom
  • Jon P Waltho, University of Manchester, United Kingdom
  • Elena Ievoli, University of Rome Tor Vergata, Italy
  • Thomas A Jowitt, University of Manchester, United Kingdom
  • Jan J Enghild
  • Ralf P Richter, CIC biomaGUNE, Spain
  • Antonietta Salustri, University of Rome Tor Vergata, Italy
  • Caroline M Milner, University of Manchester, United Kingdom
  • Anthony J Day, University of Manchester, United Kingdom

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 crosslinked network by the cooperative action of three proteins, inter-alpha-inhibitor (IalphaI), pentraxin-3 and TNF-induced protein-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. While it is known that matrix stabilization involves the TSG-6-mediated transfer of IalphaI heavy chains (HC) onto hyaluronan (to form covalent HC-HA complexes that are crosslinked 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 a HC-associated magnesium ion. In addition, we have found that the well-characterised 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 either impaired transferase and/or hyaluronan-binding functions), revealed that while 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.

Original languageEnglish
JournalJournal of Biological Chemistry
Pages (from-to)28708-28723
Number of pages16
Publication statusPublished - 27 Nov 2015

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