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J. Biol. Chem., Vol. 283, Issue 15, 10147-10161, April 11, 2008

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Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization^*

Antonio Inforzato¹², Vincenzo Rivieccio^¶1, Antonio P. Morreale, Antonio Bastone^||, Antonietta Salustri^**, Laura Scarchilli^**, Antonio Verdoliva^¶, Silvia Vincenti, Grazia Gallo, Caterina Chiapparino, Lucrezia Pacello, Eleonora Nucera, Ottaviano Serlupi-Crescenzi, Anthony J. Day, Barbara Bottazzi, Alberto Mantovani, Rita De Santis, and Giovanni Salvatori³

From the Sigma-Tau Research and Development, Pomezia 00040, Italy, Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, ^¶Tecnogen S.p.A., Piana di Monte Verna 81015, Italy, ^||Istituto Ricerche Farmacologiche "Mario Negri," Milan 20157, Italy, ^**Department of Public Health and Cell Biology, University of Rome "Tor Vergata," Rome 00133, Italy, and Istituto Clinico Humanitas (ICH), Rozzano 20089, Italy

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 Cys³¹⁷ and Cys³¹⁸ 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.

Received for publication, October 15, 2007 , and in revised form, January 24, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ Both authors contributed equally to this work.

² Supported by an international postdoctoral "Leonino Fontana and Maria Lionello" fellowship from Fondazione Italiana per la Ricerca sul Cancro (F.I.R.C.).

³ To whom correspondence should be addressed: Immunology Area, R&D Dept., Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina km 30.400, 00040 Pomezia, Rome, Italy. Tel.: 39-06-9139-3847; Fax: 39-06-9139-3988; E-mail: giovanni.salvatori{at}sigma-tau.it.

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