title: Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.
abstract: PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.
authors.affiliation: Sigma-Tau Research and Development, Via Pontina km 30.400, Pomezia, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Inforzato
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Rivieccio
authors.forename: Antonio P
authors.initials: AP
authors.lastname: Morreale
authors.forename: Antonio
authors.initials: A
authors.lastname: Bastone
authors.forename: Antonietta
authors.initials: A
authors.lastname: Salustri
authors.forename: Laura
authors.initials: L
authors.lastname: Scarchilli
authors.forename: Antonio
authors.initials: A
authors.lastname: Verdoliva
authors.forename: Silvia
authors.initials: S
authors.lastname: Vincenti
authors.forename: Grazia
authors.initials: G
authors.lastname: Gallo
authors.forename: Caterina
authors.initials: C
authors.lastname: Chiapparino
authors.forename: Lucrezia
authors.initials: L
authors.lastname: Pacello
authors.forename: Eleonora
authors.initials: E
authors.lastname: Nucera
authors.forename: Ottaviano
authors.initials: O
authors.lastname: Serlupi-Crescenzi
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
authors.forename: Barbara
authors.initials: B
authors.lastname: Bottazzi
authors.forename: Alberto
authors.initials: A
authors.lastname: Mantovani
authors.forename: Rita
authors.initials: R
authors.lastname: De Santis
authors.forename: Giovanni
authors.initials: G
authors.lastname: Salvatori
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D054885:Cumulus Cells / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D005298:Fertility / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D010060:Ovulation / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D004220:Disulfides; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins