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

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

Author information

1
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.
2
the Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
3
the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
4
the Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome 00133, Italy.
5
the Department of Molecular Chemistry, University of Aarhus, 8000 Aarhus C, Denmark.
6
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.
7
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.

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.

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

PMID:
26468290
PMCID:
PMC4661386
DOI:
10.1074/jbc.M115.669838
[Indexed for MEDLINE]
Free PMC Article

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