title: Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly.
abstract: Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N(_)PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N(_)PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IαI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N(_)PTX3 retains the ability to bind to both IαI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IαI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3 binding capacity, does not affect in vitro cumulus matrix formation thus ruling out this possibility. In conclusion, the data strength the view that PTX3 acts as a nodal molecule in cross-linking HA in the matrix.
authors.affiliation: Department of Public Health and Cell Biology, University of Rome Tor Vergata, 00133 Rome, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Ievoli
authors.forename: Ragnar
authors.initials: R
authors.lastname: Lindstedt
authors.forename: Antonio
authors.initials: A
authors.lastname: Inforzato
authors.forename: Antonella
authors.initials: A
authors.lastname: Camaioni
authors.forename: Francesca
authors.initials: F
authors.lastname: Palone
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
authors.forename: Alberto
authors.initials: A
authors.lastname: Mantovani
authors.forename: Giovanni
authors.initials: G
authors.lastname: Salvatori
authors.forename: Antonietta
authors.initials: A
authors.lastname: Salustri
mesh_terms: D000818:Animals; D001665:Binding Sites; D002097:C-Reactive Protein / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D054885:Cumulus Cells / Q000166:cytology / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000235:genetics / Q000378:metabolism*; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D015815:Cell Adhesion Molecules; D016222:Fibroblast Growth Factor 2; D006820:Hyaluronic Acid; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins; C513057:Tnfaip6 protein, mouse