title: Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component.
abstract: PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of beta-sheets with a minor alpha-helical component. While in gel filtration the protein is eluted with a molecular mass of congruent with900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca2+. Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a Kd 7.4 x 10(-8) M as determined by solid phase binding assay. The Chinese hamster ovary cell-expressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.
authors.affiliation: From the Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milano, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Bottazzi
authors.forename: V
authors.initials: V
authors.lastname: Vouret-Craviari
authors.forename: A
authors.initials: A
authors.lastname: Bastone
authors.forename: L
authors.initials: L
authors.lastname: De Gioia
authors.forename: C
authors.initials: C
authors.lastname: Matteucci
authors.forename: G
authors.initials: G
authors.lastname: Peri
authors.forename: F
authors.initials: F
authors.lastname: Spreafico
authors.forename: M
authors.initials: M
authors.lastname: Pausa
authors.forename: C
authors.initials: C
authors.lastname: D'Ettorre
authors.forename: E
authors.initials: E
authors.lastname: Gianazza
authors.forename: A
authors.initials: A
authors.lastname: Tagliabue
authors.forename: M
authors.initials: M
authors.lastname: Salmona
authors.forename: F
authors.initials: F
authors.lastname: Tedesco
authors.forename: M
authors.initials: M
authors.lastname: Introna
authors.forename: A
authors.initials: A
authors.lastname: Mantovani
mesh_terms: D000818:Animals; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D008024:Ligands; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins