title: Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.
abstract: 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.
authors.affiliation: From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: David C
authors.initials: DC
authors.lastname: Briggs
authors.affiliation: From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Holly L
authors.initials: HL
authors.lastname: Birchenough
authors.affiliation: From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Tariq
authors.initials: T
authors.lastname: Ali
authors.affiliation: the Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Marilyn S
authors.initials: MS
authors.lastname: Rugg
authors.affiliation: the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Jon P
authors.initials: JP
authors.lastname: Waltho
authors.affiliation: the Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome 00133, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Ievoli
authors.affiliation: From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jowitt
authors.affiliation: the Department of Molecular Chemistry, University of Aarhus, 8000 Aarhus C, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain, the Department of Molecular Chemistry, University Grenoble Alpes and CNRS, 38000 Grenoble, France, and the Max Planck Institute for Intelligent Systems, 70569 Stuttgart, Germany.
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Richter
authors.affiliation: the Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome 00133, Italy.
authors.forename: Antonietta
authors.initials: A
authors.lastname: Salustri
authors.affiliation: the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Milner
authors.affiliation: From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom, anthony.day@manchester.ac.uk.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054885:Cumulus Cells / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; C072273:TNFAIP6 protein, human; C513057:Tnfaip6 protein, mouse; D006820:Hyaluronic Acid
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