High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells

Rivieccio, Vincenzo; Esposito, Adelaide; Bellofiore, Piero; Palladino, Pasquale; Sassano, Marica; Colombo, Monika; Verdoliva, Antonio

doi:10.1016/j.pep.2006.07.009

Cited by 21 publications

(18 citation statements)

References 29 publications

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“…Purified Proteins and Antibodies-Recombinant human PTX3 (rhPTX3) was purified from a Chinese hamster ovary 3.5 cell line as described previously (30). Native PTX3 (nhPTX3 8387 ) was isolated from human fibrosarcoma 8387 cells (28).…”

Section: Methodsmentioning

confidence: 99%

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

Inforzato

Baldock

Jowitt

et al. 2010

Journal of Biological Chemistry

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105

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The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N-and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfidelinked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.

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Section: Methodsmentioning

confidence: 99%

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

Inforzato

Baldock

Jowitt

et al. 2010

Journal of Biological Chemistry

Self Cite

105

123

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“…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 (39). Molecular weight calibrants (thyroglobulin, ferritin, catalase, lactate dehydrogenase, and bovine serum albumin) were purchased from GE Healthcare.…”

Section: Methodsmentioning

confidence: 99%

“…Site-directed mutagenesis was performed by overlapping PCR (41). The plasmid vector pSG5 (Stratagene, La Jolla, CA) containing the full-length human PTX3 cDNA sequence (pSG5hPTX3) was used as a template (39). Overlapping primers (see Table 1) were designed using Oligo software (version 6.71) and synthesized by MWG-Biotech, Milan, Italy.…”

Section: Methodsmentioning

confidence: 99%

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

Inforzato

Rivieccio²,

Morreale

et al. 2008

Journal of Biological Chemistry

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122

129

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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.

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“…Human recombinant PTX3 (27) and rabbit polyclonal anti-PTX3 antibody were from B. Bottazzi (Humanitas Clinical Institute, Rozzano, Italy), human recombinant FGF2, ARPCA, and PARAC peptides (HPLC purity ! 95%) were from Tecnogen (Piana di Monteverna, Caserta, Italy), anti-FGFR1 antibody RR-C2 scFv and its negative control antibody a-scFv were purified as described (28).…”

Section: Reagentsmentioning

confidence: 99%

Long Pentraxin-3 Inhibits Epithelial–Mesenchymal Transition in Melanoma Cells

Ronca

Salle

Giacomini

et al. 2013

Molecular Cancer Therapeutics

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During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial-mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH 2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFRdependent tumors, including melanoma. Mol Cancer Ther; 12(12); 2760-71. Ó2013 AACR.

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High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells

Cited by 21 publications

References 29 publications

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

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

Long Pentraxin-3 Inhibits Epithelial–Mesenchymal Transition in Melanoma Cells

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