Artigo Acesso aberto Revisado por pares

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

2008; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês

10.1074/jbc.m708535200

ISSN

1083-351X

Autores

Antonio Inforzato, Vincenzo Rivieccio, Antonio 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, Giovanni Salvatori,

Tópico(s)

Biomarkers in Disease Mechanisms

Resumo

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. 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. The long pentraxin PTX3 is an acute phase glycoprotein induced in a variety of somatic and natural immunity cells by primary inflammatory stimuli (e.g. Toll-like receptor engagement, interleukin-1β, tumor necrosis factor-α, and interleukin-10) (1Breviario F. d'Aniello E.M. Golay J. Peri G. Bottazzi B. Bairoch A. Saccone S. Marzella R. Predazzi V. Rocchi M. J. Biol. Chem. 1992; 267: 22190-22197Abstract Full Text PDF PubMed Google Scholar, 2Lee G.W. Lee T.H. Vilcek J. J. Immunol. 1993; 150: 1804-1812Crossref PubMed Google Scholar, 3Alles V.V. Bottazzi B. Peri G. Golay J. Introna M. Mantovani A. Blood. 1994; 84: 3483-3493Crossref PubMed Google Scholar, 4Doni A. Michela M. Bottazzi B. Peri G. Valentino S. Polentarutti N. Garlanda C. Mantovani A. J. Leukoc. Biol. 2006; 79: 797-802Crossref PubMed Scopus (103) Google Scholar, 5Vouret-Craviari V. Matteucci C. Peri G. Poli G. Introna M. Mantovani A. Infect. Immun. 1997; 65: 1345-1350Crossref PubMed Google Scholar). Studies performed on Ptx3-null (Ptx3-/-) mice have shown that this molecule has a number of functions in vivo. PTX3 acts as a soluble pattern recognition receptor with a non-redundant protective role against selected pathogens, mainly the opportunistic fungus Aspergillus fumigatus (6Garlanda C. Hirsch E. Bozza S. Salustri A. De Acetis M Nota R. Maccagno A. Riva F. Bottazzi B. Peri G. Doni A. Vago L. Botto M. De Santis R. Carminati P. Siracusa G. Altruda F. Vecchi A. Romani L. Mantovani A. Nature. 2002; 420: 182-186Crossref PubMed Scopus (577) Google Scholar, 7Gaziano R. Bozza S. Bellocchio S. Perruccio K. Montagnoli C. Pitzurra L. Salvatori G. De Santis R. Carminati P. Mantovani A. Romani L. Antimicrob. Agents Chemother. 2004; 48: 4414-4421Crossref PubMed Scopus (122) Google Scholar). PTX3 is also essential for correct assembly of the viscoelastic hyaluronan (HA) 4The abbreviations used are:HAhyaluronanFGF-2fibroblast growth factor-2TSG-6tumor necrosis factor-stimulated gene-6 proteinCRPC-reactive proteinSAPserum amyloid P componentNP2neuronal pentraxin 2, also known as neuronal activity-regulated pentraxin (Narp)NP1neuronal pentraxin 1MALDImatrix-assisted laser desorption ionizationESIelectrospray ionizationLCliquid chromatographyMSmass spectrometryCIDcollision-induced dissociationCOCcumulus cell-oocyte complexwtwild typeCHOChinese hamster ovaryDTTdithiothreitolWBWestern blotTOFtime-of-flightHPLChigh pressure liquid chromatography.-rich matrix surrounding the oocyte in the preovulatory follicle (namely, the cumulus oophorus). In fact Ptx3-/- mice show severe female subfertility because of defective cumulus organization (8Varani S. Elvin J.A. Yan C. DeMayo J. DeMayo F.J. Horton H.F. Byrne M.C. Matzuk M.M. Mol. Endocrinol. 2002; 16: 1154-1167Crossref PubMed Scopus (269) Google Scholar, 9Salustri A. Garlanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development. 2004; 131: 1577-1586Crossref PubMed Scopus (369) Google Scholar). A number of PTX3 ligands have been described so far, suggesting different physiological and/or pathological roles for this protein. PTX3 binds with high affinity to the complement component C1q thus activating the complement system through the classical pathway (10Nauta A.J. Bottazzi B. Mantovani A. Salvatori G. Kishore U. Schwaeble W.J. Gingras A.R. Tzima S. Vivanco F. Egido J. Tijsma O. Hack E.C. Daha M.R. Roos A. Eur. J. Immunol. 2003; 33: 465-473Crossref PubMed Scopus (293) Google Scholar, 11Roumenina L.T. Ruseva M.M. Zlatarova A. Ghai R. Kolev M. Olova N. Gadjeva M. Agrawal A. Bottazzi B. Mantovani A. Reid K.B. Kishore U. Kojouharova M.S. Biochemistry. 2006; 45: 4093-4104Crossref PubMed Scopus (112) Google Scholar). The selective recognition of fibroblast growth factor-2 (FGF-2) by PTX3 inhibits FGF-2 angiogenic activity on endothelial cells (12Rusnati M. Camozzi M. Moroni E. Bottazzi B. Peri G. Indraccolo S. Amadori A. Mantovani A. Presta M. Blood. 2004; 104: 92-99Crossref PubMed Scopus (160) Google Scholar) and blocks the autocrine and paracrine stimulation exerted by FGF-2 on smooth muscle cells (13Camozzi M. Zacchigna S. Rusnati M. Coltrini D. Ramirez-Correa G. Bottazzi B. Mantovani A. Giacca M. Presta M. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1837-1842Crossref PubMed Scopus (86) Google Scholar). Previous studies on Ptx3-/- mice suggested that PTX3 acts as a central "node" in cumulus matrix organization by establishing multivalent contacts to the HA-binding protein tumor necrosis factor-stimulated gene-6 protein (TSG-6) (9Salustri A. Garlanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development. 2004; 131: 1577-1586Crossref PubMed Scopus (369) Google Scholar, 14Milner C.M. Day A.J. J. Cell Sci. 2003; 116: 1863-1873Crossref PubMed Scopus (309) Google Scholar, 15Day A.J. de la Motte C.A. Trends Immunol. 2005; 26: 637-643Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and the serum protein inter-α-inhibitor (16Scarchilli L. Camaioni A. Bottazzi B. Negri V. Doni A. Deban L. Bastone A. Salvatori G. Mantovani A. Siracusa G. Salustri A. J. Biol. Chem. 2007; 282: 30161-30170Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). PTX3 also plays a role in regulating the scavenger activity of macrophages and dendritic cells by direct binding to apoptotic cells (17Rovere P. Peri G. Fazzini F. Bottazzi B. Doni A. Bondanza A. Zimmermann V.S. Garlanda C. Fascio U. Sabbadini M.G. Rugarli C. Mantovani A. Manfredi A.A. Blood. 2000; 96: 4300-4306Crossref PubMed Google Scholar, 18van Rossum A.P. Fazzini F. Limburg P.C. Manfredi A.A. Rovere-Querini P. Mantovani A. Kallenberg C.G. Arthritis Rheum. 2004; 50: 2667-2674Crossref PubMed Scopus (91) Google Scholar, 19Baruah P. Dumitriu I.E. Peri G. Russo V. Mantovani A. Manfredi A.A. Rovere-Querini P. J. Leukoc. Biol. 2006; 80: 87-95Crossref PubMed Scopus (114) Google Scholar, 20Baruah P. Propato A. Dumitriu I.E. Rovere-Querini P. Russo V. Fontana R. Accapezzato D. Peri G. Mantovani A. Barnaba V. Manfredi A.A. Blood. 2006; 107: 151-158Crossref PubMed Scopus (91) Google Scholar). hyaluronan fibroblast growth factor-2 tumor necrosis factor-stimulated gene-6 protein C-reactive protein serum amyloid P component neuronal pentraxin 2, also known as neuronal activity-regulated pentraxin (Narp) neuronal pentraxin 1 matrix-assisted laser desorption ionization electrospray ionization liquid chromatography mass spectrometry collision-induced dissociation cumulus cell-oocyte complex wild type Chinese hamster ovary dithiothreitol Western blot time-of-flight high pressure liquid chromatography. This broad binding capacity is likely to be due to the structural complexity of the protein as compared with the classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) (21Pepys M.B. Baltz M.L. Adv. Immunol. 1983; 34: 141-212Crossref PubMed Scopus (1031) Google Scholar). Like other members of the long pentraxin subfamily, human PTX3 is composed of a pentraxin-like C-terminal domain (amino acids 179-381), which is homologous to the entire short pentraxin amino acid sequence, and an unrelated N-terminal region (amino acids 18-178) (22Goodman A.R. Cardozo T. Abagyan R. Altmeyer A. Wisniewski H.G. Vilcek J. Cytokine Growth Factor Rev. 1996; 7: 191-202Crossref PubMed Scopus (121) Google Scholar, 23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). The human protein has a unique N-glycosylation site at Asn220 that has been described to be fully occupied by complex type oligosaccharides, mainly fucosylated and sialylated biantennary sugars (24Inforzato A. Peri G. Doni A. Garlanda C. Mantovani A. Bastone A. Carpentieri A. Amoresano A. Pucci P. Roos A. Daha M.R. Vincenti S. Gallo G. Carminati P. De Santis R. Salvatori G. Biochemistry. 2006; 45: 11540-11551Crossref PubMed Scopus (102) Google Scholar). PTX3 glycosylation status affects the protein interaction with C1q thus modulating PTX3-mediated activation of the complement classical pathway (24Inforzato A. Peri G. Doni A. Garlanda C. Mantovani A. Bastone A. Carpentieri A. Amoresano A. Pucci P. Roos A. Daha M.R. Vincenti S. Gallo G. Carminati P. De Santis R. Salvatori G. Biochemistry. 2006; 45: 11540-11551Crossref PubMed Scopus (102) Google Scholar). Studies performed with recombinant isolated forms of both PTX3 C- and N-terminal domains provided preliminary information on regions of the molecule involved in ligands recognition. The C1q-binding site appears to be localized in the C-terminal domain as expected from the known interaction of both CRP and SAP with C1q (23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 25Kaplan M.H. Volanakis J.E. J. Immunol. 1974; 112: 2135-2147PubMed Google Scholar, 26Agrawal A. Scrive A.K. Greenhough T.J. Volanakis J.E. J. Immunol. 2001; 166: 3998-4004Crossref PubMed Scopus (142) Google Scholar, 27Bristol C.L. Boackle R.J. Mol. Immunol. 1986; 23: 1045-1052Crossref PubMed Scopus (52) Google Scholar, 28Ying S.C. Gewurz A.T. Jiang H. Gewurz H. J. Immunol. 1993; 150: 169-176PubMed Google Scholar). Structural determinants of the interaction with inter-α-inhibitor and FGF-2 are localized in the N-terminal domain (16Scarchilli L. Camaioni A. Bottazzi B. Negri V. Doni A. Deban L. Bastone A. Salvatori G. Mantovani A. Siracusa G. Salustri A. J. Biol. Chem. 2007; 282: 30161-30170Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 29Camozzi M. Rusnati M. Bugatti A. Bottazzi B. Mantovani A. Bastone A. Inforzato A. Vincenti S. Bracci L. Mastroianni D. Presta M. J. Biol. Chem. 2006; 281: 22605-22613Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition to the multidomain organization, PTX3 shows a complex quaternary structure with subunits assembled into high order oligomers stabilized by disulfide bonds as demonstrated by electrophoretic and chromatographic analyses (23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Emerging evidence suggests that both short and long pentraxins express distinct bioactivities depending on subunit organization. For example, native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas monomeric CRP, resulting from loss of the pentameric symmetry of the molecule, displays potent prothrombotic activities (30Khreiss T. József L. Potempa L.A. Filep J.G. Circulation. 2004; 110: 2713-2720Crossref PubMed Scopus (94) Google Scholar, 31Zouki C. Haas B. Chan J.S. Potempa L.A. Filep J.G. J. Immunol. 2001; 167: 5355-5361Crossref PubMed Scopus (75) Google Scholar). Moreover disruption of the pentameric structure, as achieved by urea treatment or by site-directed mutagenesis, leads to enhanced CRP binding to C1q and subsequent C1 activation as well as the ability of CRP to interact with complement-regulatory proteins factor H and C4b-binding protein (32Bíró A. Rovó Z. Papp D. Cervenak L. Varga L. Füst G. Thielens N.M. Arlaud G.J. Prohászka Z. Immunology. 2007; 121: 40-50Crossref PubMed Scopus (92) Google Scholar). Neuronal pentraxin 2 (NP2), also known as neuronal activity-regulated pentraxin (Narp), and neuronal pentraxin 1 (NP1), two members of the long pentraxin subfamily selectively expressed in brain, are covalently linked by disulfide bonds into highly organized complexes (33Omeis I.A. Hsu Y.C. Perin M.S. Genomics. 1996; 36: 543-545Crossref PubMed Scopus (55) Google Scholar, 34Hsu Y.C. Perin M.S. Genomics. 1995; 28: 220-227Crossref PubMed Scopus (90) Google Scholar). The number of protein molecules in these complexes is dynamically dependent upon the activity history of the neurons and the brain developmental stage (35Xu D. Hopf C. Reddy R. Cho R.W. Guo L. Lanahan A. Petralia R.S. Wenthold R.J. O'Brien R.J. Worley P. Neuron. 2003; 39: 513-528Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). However, little is known about the effect of protein oligomerization on PTX3 biological activity. It has been shown that the recombinant isolated PTX3 C-terminal domain, expressed as a monomeric species, is not able to bind C1q unless polymerized through chemical cross-linking, thus suggesting a role for the protein multimer in complement recognition possibly dependent on avidity and/or cooperativity mechanisms (23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Short pentraxins show typical pentameric symmetry with protomer subunits held together by non-covalent interactions (21Pepys M.B. Baltz M.L. Adv. Immunol. 1983; 34: 141-212Crossref PubMed Scopus (1031) Google Scholar). PTX3 C-terminal domain three-dimensional structures have been elaborated by homology modeling based on human SAP (Protein Data Bank code 1sac) and CRP (Protein Data Bank code 1b09) crystal structures (22Goodman A.R. Cardozo T. Abagyan R. Altmeyer A. Wisniewski H.G. Vilcek J. Cytokine Growth Factor Rev. 1996; 7: 191-202Crossref PubMed Scopus (121) Google Scholar, 24Inforzato A. Peri G. Doni A. Garlanda C. Mantovani A. Bastone A. Carpentieri A. Amoresano A. Pucci P. Roos A. Daha M.R. Vincenti S. Gallo G. Carminati P. De Santis R. Salvatori G. Biochemistry. 2006; 45: 11540-11551Crossref PubMed Scopus (102) Google Scholar, 36Emsley J. White H.E. O'Hara B.P. Oliva G. Srinivasan N. Tickle I.J. Blundell T.L. Pepys M.B. Wood S.P. Nature. 1994; 367: 338-345Crossref PubMed Scopus (417) Google Scholar, 37Thompson D. Pepys M.B. Wood S.P. Struct. Fold. Des. 1999; 7: 169-177Abstract Full Text Full Text PDF Scopus (577) Google Scholar, 38Introna M. Alles V.V. Castellano M. Picardi G. De Gioia L. Bottazzi B. Peri G. Breviario F. Salmona M. De Gregorio L. Dragani T.A. Srinivasan N. Blundell T.L. Hamilton T.A. Mantovani A. Blood. 1996; 87: 1862-1872Crossref PubMed Google Scholar). Most interestingly, the pattern of amino acid residues located at the protomer interface in SAP pentamers is not conserved in PTX3, suggesting that PTX3 quaternary assembly might not fit the characteristic short pentraxin pentameric arrangement (38Introna M. Alles V.V. Castellano M. Picardi G. De Gioia L. Bottazzi B. Peri G. Breviario F. Salmona M. De Gregorio L. Dragani T.A. Srinivasan N. Blundell T.L. Hamilton T.A. Mantovani A. Blood. 1996; 87: 1862-1872Crossref PubMed Google Scholar). In contrast to CRP and SAP, PTX3 oligomers are stabilized by covalent bonds (23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). The mature protein contains nine cysteine residues: three are located within the N-terminal region (Cys47, Cys49, and Cys103), and six are in the C-terminal domain (Cys179, Cys210, Cys271, Cys317, Cys318, and Cys357). Cys residues at positions 210 and 271 are highly conserved among pentraxins and based on the homology with CRP and SAP are predicted to be engaged in an intrachain disulfide bond (22Goodman A.R. Cardozo T. Abagyan R. Altmeyer A. Wisniewski H.G. Vilcek J. Cytokine Growth Factor Rev. 1996; 7: 191-202Crossref PubMed Scopus (121) Google Scholar, 23Bottazzi B. Vouret-Craviari V. Bastone A. De Gioia L. Matteucci C. Peri G. Spreafico F. Pausa M. D'Ettorre C. Gianazza E. Tagliabue A. Salmona M. Tedesco F. Introna M. Mantovani A. J. Biol. Chem. 1997; 272: 32817-32823Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). In this study we resolved the recombinant PTX3 oligomeric assembly, determining the number of subunits forming the functional protein. Both SDS-PAGE, performed under non-reducing conditions, and native-PAGE, carried out according to the Ferguson plot method, showed PTX3 to form mainly covalent octamers. We then identified the network of disulfide bonds stabilizing PTX3 multimers by exploiting two complementary approaches: mass spectrometry and Cys to Ser site-directed mutagenesis. Based on data from both techniques a topological map of PTX3 interchain disulfide bonds was drawn where the protein molecule is represented as a covalent octamer composed of two equivalent tetramers. The contribution of disulfide bonds to protein oligomerization was assessed by PAGE analysis under non-denaturing conditions. The new structural constraints were used to build a refined model of the PTX3 C-terminal domain. A previous study showed that cumuli from Ptx3-deficient mice are unable to organize the glycosaminoglycan HA in a stable matrix and that exogenously added recombinant PTX3 rescues defective cumulus expansion ex vivo (9Salustri A. Garlanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development. 2004; 131: 1577-1586Crossref PubMed Scopus (369) Google Scholar, 16Scarchilli L. Camaioni A. Bottazzi B. Negri V. Doni A. Deban L. Bastone A. Salvatori G. Mantovani A. Siracusa G. Salustri A. J. Biol. Chem. 2007; 282: 30161-30170Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We report here that the rescue activity of PTX3 mutants, forming tetramers instead of octamers, was comparable to that of the wt protein. PTX3 mutants, lacking the entire set of interchain disulfide bonds and mainly composed of dimers, proved less effective than the wt protein in rescuing defective cumulus expansion. PTX3 oligomerization is thus essential to protein functionality within the cumulus matrix. Moreover the two tetramers composing the native PTX3 octamer appear to act as functional units in cumulus matrix organization. These findings showed that PTX3 multimeric status plays a key role in defining the biological activity of the protein. Purified Proteins and Antibodies—Human recombinant PTX3 was purified from a CHO 3.5 cell line stably and constitutively expressing the protein as described previously (39Rivieccio V. Esposito A. Bellofiore P. Palladino P. Sassano M. Colombo M. Verdoliva A. Protein Expr. Purif. 2007; 51: 49-58Crossref PubMed Scopus (22) Google Scholar). Molecular weight calibrants (thyroglobulin, ferritin, catalase, lactate dehydrogenase, and bovine serum albumin) were purchased from GE Healthcare. Biotin-labeled rabbit anti-human PTX3 polyclonal antibody αPTX3pb and rat anti-human PTX3 monoclonal antibody MNB4 were obtained from "Mario Negri" Pharmacological Research Institute, Milan, Italy. Horseradish peroxidase-conjugated streptavidin was from GE Healthcare, and rabbit anti-rat IgG-horseradish peroxidase conjugate was from Dako, Glostrup, Denmark. Enzymes and Chemicals—Proteomics grade trypsin (from porcine pancreas), α-chymotrypsin (Nα-p-tosyl-l-lysine chloromethyl ketone-treated from bovine pancreas), endoproteinase Asp-N (from Pseudomonas fragi mutant strain), and endoproteinase Glu-C (from Staphylococcus aureus V8) were purchased from Sigma-Aldrich. DNA Taq polymerase and restriction enzymes applied in molecular cloning were from Takara Bio, Otsu, Japan. Tris acetate 3-8% gradient gels were obtained from Invitrogen. Other electrophoresis reagents and nonfat dry milk were from Bio-Rad. Hybond-C Extra nitrocellulose membranes and ECL Plus Western blotting detection reagents were from GE Healthcare. Phosphate-buffered saline tablets, Tris base, NH4HCO3, NaCl, Tween 20, dithiothreitol (DTT), iodoacetamide, and α-cyano-4-hydroxycinnamic acid were purchased from Sigma-Aldrich. All other reagents and solvents were from Carlo Erba, Rodano, Italy and were of the highest purity available. Ferguson Plot—Aliquots of purified recombinant PTX3 (5 μg/lane) were loaded on homogeneous polyacrylamide gels cast at 5.0, 5.5, 6.0, and 6.5% (w/v) total acrylamide concentration (% T) in the absence of SDS. Runs were performed under non-denaturing conditions in 50 mm Tris-HCl, 200 mm glycine, pH 8.3, at 150-V constant voltage for 1 h, and proteins were detected by Coomassie staining. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa) were used as molecular mass calibrants. Protein mobility was analyzed by Ferguson plot as described previously (40Butterman M. Tietz D. Orbán L. Chrambach A. Electrophoresis. 1988; 9: 293-298Crossref PubMed Scopus (27) Google Scholar). Briefly the relationship between protein mobility (μ) and sieving matrix concentration (% T) may be written as log10(μ)=log10(μ0)-Kr(%T)(Eq. 1) where μ0 is the free solution mobility (zero concentration of sieving matrix) and Kr is the retardation coefficient (which is directly related to protein shape and molecular weight). A standard curve (namely the Ferguson plot) was constructed by plotting the logarithm of calibrant relative mobility (log10Rf) as a function of total polyacrylamide concentration (% T). Retardation coefficients (Kr) were then computed from the plot slope and graphed versus molecular mass (Kr plot). PTX3 samples were processed in the same way as the calibrants, and their molecular mass was finally calculated by Kr plot interpolation. In a separate set of experiments, electrophoresis mobility data for Ferguson plot analysis of purified recombinant PTX3 were derived from Western blots (WBs). Briefly aliquots of protein (20 ng/lane) were loaded on native gels and run under the same experimental conditions as above. Gels were then blotted onto nitrocellulose membranes, revealed by immunodetection (see below), and submitted to Ferguson plot analysis as described above. SDS-PAGE and WB—Aliquots of purified recombinant PTX3 (either 2 μg for Coomassie staining or 20 ng for immunodetection) were resolved on Tris acetate 3-8% gradient or Tris-glycine 9% homogeneous gels under denaturing conditions in the presence or absence of DTT. Following electrophoresis, proteins were either revealed by Coomassie staining or transferred to Hybond-C Extra nitrocellulose membranes for subsequent immunodetection. Blotted membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% (v/v) Tween 20 (blocking buffer) for 2 h at room temperature and then incubated overnight at 4 °C with a 1:2,000 MNB4 dilution in blocking buffer. This antibody recognizes a linear epitope mapping in the N-terminal domain of human PTX3 (29Camozzi M. Rusnati M. Bugatti A. Bottazzi B. Mantovani A. Bastone A. Inforzato A. Vincenti S. Bracci L. Mastroianni D. Presta M. J. Biol. Chem. 2006; 281: 22605-22613Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Following an additional incubation with rabbit anti-rat IgG-horseradish peroxidase conjugate, membranes were finally developed with ECL Plus according to the manufacturer's instructions. PTX3 Proteolysis and MALDI-MS—Purified recombinant PTX3 in phosphate-buffered saline (1 mg/ml) was dialyzed against 100 mm NH4HCO3, pH 7.8, by ultrafiltration on Microcon YM-30 centrifugal filter units (Millipore, Billerica, MA). Protein samples (200 μg) were subjected to proteolysis by incubation with trypsin, α-chymotrypsin, Asp-N, or Glu-C endoproteinases. Digestions were performed in 100 mm NH4HCO3, pH 7.8, at 37 °C for 16 h in a 225-μl final volume using enzyme: protein ratios of 1:50, 1:100, 1:50, and 1:40 (w/w), respectively. Reactions were terminated by addition of 25 μl of 2% (v/v) trifluoroacetic acid and lyophilization. PTX3 peptide mixtures were then resuspended in 100 mm NH4HCO3, pH 7.8, and split in 50-μg aliquots. Some aliquots were directly submitted to MS analysis; others were first reduced and/or alkylated by incubation with 1 mm DTT for 1 h at 37 °C and/or 20 mm iodoacetamide for an additional 30 min at room temperature. Prior to MALDI-MS analysis, peptide mixtures from endoproteinase digestions were desalted by reversed phase chromatography on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA). Briefly peptide samples were loaded on a Phenomenex Jupiter C18 column (500 μm; 2.0-mm inner diameter × 15-cm length; particle size, 300 Å; Phenomenex, Torrance, CA) equilibrated by a 95% solvent A, 5% solvent B mixture (solvent A: 0.1% (v/v) trifluoroacetic acid; solvent B: 95% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid). Following an isocratic washing step at 5% solvent B for 10 min, a fast linear gradient was applied from 5 to 95% solvent B over 1 min, and peptide mixtures were eluted at 95% solvent B. The flow rate was set to 0.2 ml/min, and peptide elution was monitored as UV absorbance at both 216 and 280 nm. Peptide mixtures, eluted as a single peak, were manually collected and immediately freezedried. Positive ion MALDI-MS analyses of PTX3 peptides were carried out on a Bruker Daltonics Reflex II TOF/TOF MALDI spectrometer (Bruker Daltonics, Billerica, MA) operating in reflectron mode. The MALDI matrix was prepared by dissolving 10 mg of α-cyano-4-hydroxycinnamic acid in 1 ml of acetonitrile, 0.2% (v/v) trifluoroacetic acid (70:30, v/v). Typically 1 μl of matrix was applied to the metallic sample plate, and 1 μl of analyte was then added. Acceleration and reflector voltages were set as follows: target vol

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