期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 290, 期 48, 页码 28708-28723出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.669838
关键词
hyaluronan; protein structure; protein-protein interaction; reproduction; site-directed mutagenesis; CUB module structure; TSG-6; cumulus-oocyte complex expansion; heavy chain-hyaluronan complex formation; inter--inhibitor
资金
- Arthritis Research Campaign [14871, 18472, 19489]
- Medical Research Council [G0701180]
- Biotechnology and Biological Sciences Research Council CASE award
- Biotechnology and Biological Sciences Research Council [957322, BB/H000844/1] Funding Source: researchfish
- Medical Research Council [G0701180, MC_U138274352] Funding Source: researchfish
- Versus Arthritis [18472, 19489] Funding Source: researchfish
- BBSRC [BB/H000844/1] Funding Source: UKRI
- MRC [G0701180, MC_U138274352] Funding Source: UKRI
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--inhibitor (II), 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 II heavy chains (HCs) onto hyaluronan (to form covalent HCHA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HCTSG-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 HCTSG-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 HCHA 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.
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