TY - JOUR
T1 - Metal Ion-Dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix
AU - Briggs, David C
AU - Birchenough, Holly L
AU - Ali, Tariq
AU - Rugg, Marilyn S
AU - Waltho, Jon P
AU - Ievoli, Elena
AU - Jowitt, Thomas A
AU - Enghild, Jan J
AU - Richter, Ralf P
AU - Salustri, Antonietta
AU - Milner, Caroline M
AU - Day, Anthony J
N1 - Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
PY - 2015/11/27
Y1 - 2015/11/27
N2 - 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.
AB - 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.
U2 - 10.1074/jbc.M115.669838
DO - 10.1074/jbc.M115.669838
M3 - Journal article
C2 - 26468290
SN - 0021-9258
VL - 290
SP - 28708
EP - 28723
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
ER -