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Multimer Formation and Ligand Recognition by the Long Pentraxin PTX3

Multimer Formation and Ligand Recognition by the Long Pentraxin PTX3 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 52, Issue of December 26, pp. 32817–32823, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 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* (Received for publication, August 6, 1997, and in revised form, October 6, 1997) Barbara Bottazzi‡§, Vale ´ rie Vouret-Craviari‡§, Antonio Bastone‡§, Luca De Gioia‡, Cristian Matteucci‡, Giuseppe Peri‡, Fabio Spreafico‡, Mario Pausa¶, Cinzia D’Ettorrei, Elisabetta Gianazza**, Aldo Tagliabuei, Mario Salmona‡, Francesco Tedesco‡‡, Martino Introna‡, and Alberto Mantovani‡§§¶¶ ‡From the Istituto di Ricerche Farmacologiche “Mario Negri,” Via Eritrea 62, 20157 Milano, Italy; ¶Istituto di Ostetricia e Ginecologia, IRCCS Burlo Garofolo, Trieste, Italy; iDOMPE’ S.P.A., L’Aquila, Italy; **Istituto di Scienze Farmacologiche, Universita ` di Milano, Milan, Italy; ‡‡Dipartimento di Fisiologia e Patologia, Universita ` di Trieste, Trieste, Italy; and §§Sezione di Patologia e Immunologia, Dipartimento di Biotecnologie, Universita ` di Brescia, Brescia, Italy PTX3 is a prototypic long pentraxin consisting of a quirement for disulfide bonds, and does not recognize C-terminal 203-amino acid pentraxin-like domain cou- CRP/SAP ligands. The capacity to bind C1q, mediated by pled with an N-terminal 178-amino acid unrelated por- the pentraxin domain, is consistent with the view that tion. The present study was designed to characterize the PTX3, produced in tissues by endothelial cells or macro- structure and ligand binding properties of human PTX3, phages in response to interleukin-1 and tumor necrosis in comparison with the classical pentraxins C-reactive factor, may act as a local regulator of innate immunity. protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 Pentraxins (C-reactive protein, CRP, and serum amyloid P (Glu). Lectin binding and treatment with N-glycosidase component, SAP) are acute phase proteins conserved during F showed that PTX3 is N-glycosylated, sugars account- evolution from Limulus polyphemus to man (1– 6). Pentraxins ing for 5 kDa of the monomer mass (45 kDa). Circular are composed of monomers with a b-jelly roll topology that dichroism analysis indicated that the protein consists usually assemble into pentameric, noncovalently associated b-sheets with a minor a-helical com- predominantly of structures (7–9). CRP and SAP are made in the liver in re- ponent. While in gel filtration the protein is eluted with sponse to inflammatory mediators, most prominently interleu- a molecular mass of >900 kDa, gel electrophoresis using kin-6 (10, 11). A number of ligands, recognized in a calcium- nondenaturing, nonreducing conditions revealed that dependent manner, have been identified for CRP and SAP, PTX3 forms multimers predominantly of 440 kDa appar- including phosphoethanolamine (PE), phosphocholine (PC) ent molecular mass, corresponding to decamers, and (12), DNA and chromatin (13–15), immune complexes, various that disulfide bonds are required for multimer forma- sugars (16), the best characterized of which is methyl 4,6-O-(1- tion. The ligand binding properties of PTX3 were then carboxyethylidene)-b-D-galactopyranoside (MObDG) (17), and examined. As predicted based on modeling, inductive complement components (13, 18 –22). Moreover, SAP binds to coupled plasma/atomic emission spectroscopy showed all forms of amyloid fibrils (23) and, in addition, to fibronectin, . Unlike the that PTX3 does not have coordinated Ca C4-binding protein (24, 25), and glycosaminoglycans (26). Pen- classical pentraxins CRP and SAP, PTX3 did not bind traxins represent a mechanism of innate resistance against phosphoethanolamine, phosphocholine, or high pyru- vate agarose. PTX3 in solution, bound to immobilized microbes, tools to scavenge cellular debris and components of C1q, but not C1s, and, reciprocally, C1q bound to immo- the extracellular matrix as illustrated by amyloid deposits (3). bilized PTX3. Binding of PTX3 to C1q is specific and PTX3 is a prototypic long pentraxin, structurally related to, 7.4 3 10 M as determined by solid saturable with a K d yet distinct from, classical pentraxins. PTX3 was cloned as an phase binding assay. The Chinese hamster ovary cell- interleukin-1-inducible gene in endothelial cells (27) and as a expressed pentraxin domain bound C1q when multim- tumor necrosis factor-inducible gene (TSG 14) in fibroblasts erized. Thus, as predicted on the basis of computer mod- (28). PTX3 is constituted of a C-terminal pentraxin-like domain eling, the prototypic long pentraxin PTX3 forms of 203 amino acids encoded by the third exon and by an N- multimers, which differ from those formed by classical terminal 178- amino acid long portion (27). Inflammatory cy- pentraxins in terms of protomer composition and re- tokines induce PTX3 expression in a variety of cell types, most prominently endothelial cells and mononuclear phagocytes (27, 29 –31). After cloning of PTX3, other “long pentraxins” were * This work was supported by the Associazione Italiana Ricerca sul identified, including guinea pig apexin (32, 33), XL-PXN1 from Cancro (AIRC), by the special project Biotechnology, Consiglio Nazion- ale delle Ricerche (CNR), by Istituto Superiore di Sanita ` (project On- cology), and by Ministero della Ricerca Scientifica e Tecnologica (40 and 60%), Italy. The costs of publication of this article were defrayed in part The abbreviations used are: CRP, C-reactive protein; SAP, serum by the payment of page charges. This article must therefore be hereby amyloid P component; PE, phosphoethanolamine; PC, phosphocholine; marked “advertisement” in accordance with 18 U.S.C. Section 1734 MObDG, methyl 4,6-O-(1-carboxyethylidene)-b-D-galactopyranoside; solely to indicate this fact. PBS, phosphate-buffered saline; HPA, high pyruvate agarose; NP, neu- § These authors have contributed equally to this work. ronal pentraxin; bp, base pair(s); CHO, Chinese hamster ovary; PAGE, ¶¶ To whom correspondence should be addressed: Fax: 39/2/3546277; polyacrylamide gel electrophoresis; DTT, dithiotreitol; RU, resonance E-mail: [email protected]. units. This paper is available on line at http://www.jbc.org 32817 This is an Open Access article under the CC BY license. 32818 Characterization of the Long Pentraxin PTX3 pH 7.4, 1% Triton X-100, 0.1% SDS, and 1 unit of N-glycosidase F Xenopus (34), rat neuronal pentraxin (NP) (35), human neuro- (Boehringer Mannheim GmbH, Mannheim, Germany). After overnight nal pentraxin (NPTX2) (36), which possibly represents the incubation at room temperature, samples were analyzed by SDS-PAGE. human homologue of apexin, and Narp (37), which possibly Purification and Cross-linking of sPTX3—Supernatant from sPTX3 represents the rat homologue of apexin. In all these molecules cells was concentrated by ultrafiltration, and the buffer was changed to a C-terminal pentraxin domain is coupled to diverse, unrelated 50 mM Tris-HCl, pH 7, before application on a HR 5/5 Mono Q column. N-terminal portions (32–36). The structure and function of long sPTX3 was eluted with a linear gradient of NaCl (from 0 to 1 M)in50 mM Tris-HCl. Fractions recognized by the 16B5 monoclonal antibody pentraxins is unknown. were pooled, concentrated by ultrafiltration, and subjected to cross- The present study was designed to express the prototypic linking. sPTX3 (200 mgin200 ml of Veronal-buffered saline) was cross- long pentraxin PTX3 in an eukaryotic system and to charac- linked by adding 20 mlof10mM bis(sulfosuccinimidyl)suberate (Pierce) terize its structure and ligand recognition in comparison to for1hat room temperature. Then 40 ml of Tris-buffered saline was classical pentraxins. In particular, experiments were designed added to stop the reaction. to test predictions made on the basis of modeling of the pen- Ligand Binding Assays—Binding of PTX3 with Sepharose-immobi- lized PE, Sepharose-immobilized PC, or high piruvate agarose (HPA) traxin domain of PTX3 on the three-dimensional structure of was performed as described previously for CRP and SAP (41). The SAP (31). presence of PTX3 was assayed by Western blot, and results are ex- pressed as area under the curve after densitometric analysis of the EXPERIMENTAL PROCEDURES exposed film performed by the scanning densitometer GS300 (Hoefer Production of Recombinant PTX3—A 1311-bp fragment of human Scientific Instrument, San Francisco, CA). As a control, acute phase PTX3 cDNA, containing the complete coding sequence, was subcloned human serum was incubated in parallel with immobilized ligand and in pSG5 vector (Stratagene, La Jolla, CA) and transfected in CHO cells processed as for PTX3, then assayed for the presence of CRP and/or SAP by calcium phosphate precipitation. Two clones selected with G418 (Life by electroimmunoassay as described previously (41). Technologies, Inc., Paisley, Scotland, UK) were used in the present Binding of PTX3 to C1q was performed as described previously for study, CHO 3.5, producing high levels of PTX3, and CHO 2.1, trans- CRP and SAP (19, 20). Briefly 96-well plates were coated with 250 –500 fected with the antisense construct. Conditioned medium was collected ng of C1q (Calbiochem) in PBS with calcium and magnesium (4 °C from confluent monolayers incubated 24 h with culture medium (Dul- overnight). Wells were washed with PBS plus 0.05% Tween 20, blocked becco’s modified Eagle’s medium; Seromed, Berlin, Germany) without with 0.5% dry milk in PBS (2 h at room temperature), and extensively fetal calf serum. The pentraxin domain of PTX3 was obtained by intro- washed before the addition of 100 ml of supernatant from 3.5 or 2.1 cell duction by polymerase chain reaction of a XhoI site at the end of the lines or 200 ng of purified PTX3 diluted in PBS (30 min, 37 °C). After signal peptide (position 18 of the published sequence) (27) and at the 59 washing, plates were incubated with 16B5 monoclonal antibody (1 h at end of the pentraxin domain (position 172). The fragment was sub- room temperature), washed again, and incubated with horseradish cloned in pSG5 vector and transfected in CHO cells by calcium phos- peroxidase-labeled goat anti-rat IgG (Amersham; 1:2000;1hat room phate precipitation as described above. A high recombinant producer temperature). After extensive washing, 100 ml of chromogen substrate clone, named sPTX3, was selected. ABTS were added (Kirkegaard and Perry, Gaithersburg, MD), and Gel Electrophoresis and Western Blot Analysis—5–10% polyacryl- absorbance values were read at 405 nm. As positive control for the amide gradient gel in the presence of SDS was run in the discontinuous binding assay, immobilized rabbit IgG heated at 63 °C for 20 min buffer system of Laemmli (38). Gels were stained with Amido Black, immediately prior to use was used (Agg-IgG) (42). Coomassie Brilliant Blue, or silver nitrate (39). For Western blots, In another set of experiments, wells were coated by overnight incu- separated proteins were electroblotted onto nitrocellulose filters (Hy- bation at 4 °C with 200 ml of purified PTX3 (2.5–10 mg/ml) in 100 mM bond ECL, Amersham Corp.) and labeled with anti-PTX3 monoclonal sodium carbonate, pH 9.6. After blocking, C1q was added in amounts antibody (see below) following standard procedures. Labeled proteins varying between 0.125 and 2 mg/ml, and incubation was continued for were detected by enhanced chemiluminescence (ECL, Amersham Corp.) 1 h at 37 °C. The bound C1q was revealed by its reaction with a specific in accordance with the manufacturer’s instructions. PTX3 was analyzed rabbit antibody for1hat37 °C followed, after washing, by biotin- in the native state in 5–10% gradient polyacrylamide gel electrophore- labeled goat anti-rabbit IgG for an additional h at 37 °C. The wells were sis (PAGE). washed and incubated with 200 ml of alkaline phosphatase-conjugated Protein Purification—Culture supernatant from CHO 3.5 cells was streptavidin (Jackson Laboratories, West Grove, PA) diluted 1/8000 for concentrated, and the buffer was changed to 50 mM imidazole, pH 6.6, 30 min at 37 °C. The enzymatic reaction was developed using the before application on a HR 5/5 Mono Q column (Pharmacia Biotech, substrate p-nitrophenyl phosphate (Sigma; 1 mg/ml) in 0.1 M glycin Uppsala, Sweden) preequilibrated with the same buffer. The column buffer pH 10.4 containing 0.1 mM MgCl and 0.1 mM ZnCl . In some 2 2 was washed until the absorbance was stable, and PTX3 was eluted with experiments C1q purified from human plasma following the procedure 1 M NaCl in 50 mM imidazole at 1 ml/min using a nonlinear gradient. In originally described by Tenner et al. (43) was used with similar results. the first step, the NaCl concentration was increased from 0% to 58% in Binding of PTX3 to C1q was characterized using biotin-labeled pu- 35 min. Then the NaCl concentration was immediately increased to rified PTX3 (bPTX3). Concentrations ranging from 25 to 800 ng of 100%, and PTX3 was obtained as a narrow peak as monitored by UV bPTX3 (0.56 –17.92 pmol considering a molecular mass of 45 kDa for detection at 280 nm. The PTX3-containing fraction was subjected to gel PTX3 monomer) in 100 ml were added to triplicate wells coated with 200 filtration on Sephacryl S-300, and PTX3 was finally eluted with PBS. ng of C1q (0.49 pmol). After 1 h incubation at 37 °C, bound PTX3 was The purification of PTX3 was monitored by SDS-PAGE and Western detected with horseradish peroxidase-labeled avidin (1/2000, 1 h) and blot analysis. To further control the elution profile, purified PTX3 was chromogen substrate ABTS, as described. The results were converted to applied to a Superose 6 column (Pharmacia Biotech) calibrated with picomolar concentration using a standard curve of bPTX3 and consid- molecular weight standards and eluted with PBS at a flow rate of 0.4 ering a molecular mass of 45 kDa for PTX3 monomer. K and B were d max ml/min. Elution was monitored by UV detection at 280 nm, and frac- obtained by nonlinear fitting of the saturation curves (44). Binding of tions (2 min each) were subsequently analyzed on native PAGE. bPTX3 to type IV collagen (Sigma; 10 mg/ml), fibronectin (Sigma; 10 Antibodies—A rat (16B5) and mouse (1C8) antibody against human mg/ml), and gelatin (Sigma; 0.5%) was analyzed with essentially the PTX3 were used in this study. Rabbit antiserum to human C1q was same protocol. purchased from Istituto Behring SpA (Scoppito, Italy), and biotin-la- Biosensor Analysis—The interaction of PTX3 with C1q was also beled goat anti-rabbit IgG was obtained from Sigma. analyzed with the BIAcore® system (biomolecular interaction analysis; Lectin Staining and Deglycosylation—Purified PTX3 (5 mg) after BIAcore AB, Uppsala, Sweden). C1q was immobilized on a CM5 sensor SDS-PAGE and electroblotting was incubated with the following lectin- chip (Pharmacia Biosensor) using the amine coupling kit (Pharmacia peroxidase conjugates at a concentration of 50 mg/ml: concanavalin A, Biosensor) (45). A volume of 30 ml of ligand (10 mg/ml C1q in 10 mM wheat germ agglutinin, Bandeiraea simplicifolia lectin BS-II, Vicia sodium acetate, pH 4.1) was immobilized with a continuous flow of HBS faba lectin, Psophocarpus tetragonolobus lectin, and Tetragonolobus (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% BIAcore® purpureas lectin. The zymogram for peroxidase was developed accord- surfactant P20, pH 7.4) at 5 ml/min. Each binding assay was performed ing to Taketa (40) in a solution containing 2 mg/ml NADH, 0.6 mg/ml with a constant flow rate (5 ml/min) of HBS, pH 7.4 at 25 °C. The nitro blue tetrazolium, 0.4 mg/ml phenol, and 3 ml/ml H O , in phos- analyte PTX3 was injected over the ligand surface in HBS and the 2 2 phate buffer, pH 7.0. Purified PTX3 (160 mg in PBS) was first made 1% surface was then regenerated by injection of 5 mlof25mM NaOH. in SDS and incubated at 100 °C for 5 min, then diluted 5-fold with Analyte binding was calculated as the difference in RU before and after concentrated buffer to a final concentration of 50 mM sodium phosphate, the interaction of ligand with analyte. For saturation analysis, PTX3 Characterization of the Long Pentraxin PTX3 32819 siderable donor to donor variation (29). To test for the pres- ence of oligosaccharides, PTX3 was stained with different lectin-peroxidase conjugates. PTX3 showed a strong staining for peroxidase-conjugated concanavalin A (specific for a-D-man- nose and a-D-glucose) and wheat germ agglutinin (specific for (D-GlcNAc)2NeuAc, Fig. 1, panel B) while it was not stained by B. simplicifolia, V. faba, P. tetragonolobus, and T. purpureas (data not shown). To better characterize the glycosylation of PTX3, the purified protein was treated with N-glycosidase F, an enzyme that cleaves Asn-linked high mannose as well as hybrid and com- plex oligosaccarides, and the deglycosylation was monitored as FIG.1. Purification and glycosylation of PTX3 expressed in CHO cells. Panel A, proteins were separated on 4 –10% SDS-PAGE and an increase in electrophoretic mobility. As shown in Fig. 1, analyzed by Western blotting (lanes 1 and 2) and silver staining (lane panel C, after treatment with N-glycosidase F, PTX3 exhibited 3). Lane 1, supernatant from CHO 3.5 cells; lanes 2 and 3, the same a decrease of approximately 5 kDa, from 45 to 40 kDa. It is after purification. Panel B, purified protein was run on 7.5–17.5% concluded that PTX3 is glycosylated and that N-linked sugars SDS-PAGE and stained with Amido Black (lane 1), concanavalin A (lane 2), or wheat germ agglutinin (lane 3). Panel C, effect of enzymatic account for 5 kDa of the major 45-kDa PTX3 protomer. Isoelec- deglycosylation on PTX3. Purified PTX3 was denatured by SDS and trofocusing of purified PTX3 showed five different bands with a incubated in the absence (lane 1) or presence of N-glycosidase F (lane 2) pI ranging from 4.5 to 4.65 (data not shown), possibly indicat- overnight at room temperature. Control and enzyme-treated samples ing heterogeneity in glycosylation in agreement with a previous were analyzed by Western blot after SDS-PAGE. The molecular mass standards (3 10 ) are indicated on the left. suggestion (29). Conformation and Multimer Formation—The purified mate- rial was used to generate a CD spectrum to determine the was serially diluted in HBS to concentrations ranging from 20 to 600 possible conformation of the protein. The spectrum is charac- nM. Each sample (30 ml) was injected and allowed a total contact time terized by a positive band at 199 nm and a negative band at 217 with the ligand surface of 6 min. nm. Careful observation of the negative band shows the pres- ence of shoulders at 209 and 224 nm. Spectral deconvolution RESULTS indicates a predominantly b-sheet protein, with some contri- Production and Purification of Human Recombinant PTX3— bution of a-helical structure (data not shown). The culture supernatant of the CHO 3.5 cell line was analyzed Classical pentraxins form noncovalently linked multimers by SDS-PAGE under reducing conditions, and a protein with (7–9). In an effort to obtain indications as to the capacity of an apparent molecular mass of 45 kDa, which was not present PTX3 to form multimers, purified protein was analyzed by gel in the culture supernatant from the antisense clone 2.1 (not filtration on Superose 6. As shown in Fig. 2, panel A,ongel shown), was observed. Western blot analysis of the same cul- filtration PTX3 eluted with an apparent molecular mass of ture supernatant showed that this protein is recognized by the about 900 kDa (similar results were obtained also with monoclonal anti-PTX3 antibody 16B5 (Fig. 1, panel A, lane 1). Sephacryl S300). When fractions from gel filtration containing To purify the protein, 500 ml of culture supernatant from the protein were run on a native gel, a pattern similar to that CHO 3.5 cells were collected, concentrated by ultrafiltration, shown in Fig. 2, panels D and E (see below) was observed. and then subjected to ion exchange chromatography on a Mono To further characterize the PTX3 multimers, denaturation with SDS and reduction with DTT were used. The apparent Q column. The fractions containing PTX3 were subsequently size of the PTX3 multimers was determined by gel electro- subjected to gel filtration in PBS. The process of purification phoresis followed by staining or Western blotting. In the ab- was monitored by SDS-PAGE and by microsequence analysis sence of reducing agents, SDS treatment caused disassembly of (see below). The purification scheme yielded PTX3 prepara- the multimers formed by SAP (not shown) and CRP (Fig. 2, tions of considerable purity (Fig. 1, panel A, lanes 2 and 3) with panel B), to their constituent monomers. In contrast, under the an occasional 66-kDa contaminant. About 5 mg of pure PTX3 same conditions, SDS treatment in the absence of DTT did not were routinely obtained from 500 ml of conditioned medium. disaggregate PTX3, which migrated as two high molecular Microsequence analysis, performed on two occasions on the N mass bands that barely entered the gel (Fig. 2, panels B and C). terminus of the purified protein, confirmed the purity and SAP multimers are clearly visible under nondenaturing, non- showed identity with the amino acid sequence predicted on the reducing conditions as a major 230-kDa band (Fig. 2, panel D). basis of the cDNA (Glu-Asn-Ser-Asp-Asp-Tyr-Asp-Leu-Met- Under the same native conditions PTX3 migrates in the gel as Tyr-Val-Asn-Leu-Asp-Asn-Glu-Ile); it demonstrates that the a predominant 440-kDa apparent molecular mass species, pos- predicted leader peptide is removed when the protein is se- sibly corresponding to a decamer, as revealed by direct staining creted, the first sequenced amino acid being Glu of the pub- and Western blotting (Fig. 2, panels D and E). In addition, two lished sequence (27, 31). minor higher forms in the 540 – 600 kDa range were usually Glycosylation—The amino acid sequence predicts a molecu- visible. This relative distribution of different multimeric forms lar mass for the reduced protein of 40 kDa instead of the 45 was observed both in silver-stained gel and in Western blot kDa observed in SDS-PAGE. The PTX3 sequence shows the (Fig. 2, panel D and E) and is not dependent on concentration, presence of a potential N-linked glycosylation site at amino since gel filtration and PAGE performed with diluted PTX3 (up acid position 220 (27). SDS-PAGE of the reduced protein shows to the sensitivity of these assays) gave similar results (not the presence of two closely related bands (Fig. 1, panels A and shown). When PTX3 was incubated with increasing concentra- C). The lower band has a calculated molecular mass of 40 kDa, tions of DTT prior to SDS-PAGE, progressively smaller aggre- the size expected for PTX3 on the basis of the amino acidic gates were observed until, at 1 mM DTT, all protein ran in the sequence without the leader peptide. The amount of 40-kDa form of the 45-kDa protomer (not shown). material was variable from preparation to preparation. It is noteworthy that the same two bands were present in superna- tants of stimulated endothelial cells and monocytes, with con- N. Polentarutti and M. Introna, unpublished results. 32820 Characterization of the Long Pentraxin PTX3 Binding of PTX3 to C1q was also observed when soluble C1q was tested on immobilized PTX3 (Fig. 4, panel C). To characterize the binding of PTX3 to C1q, serial dilutions of biotinylated PTX3 were added to immobilized C1q and the amount of bound PTX3 evaluated on the basis of a standard curve of the biotinylated protein. Fig. 4, panel D, represents a typical experiment showing the binding of PTX3 to C1q; the binding is saturable with a K of 7.4 3 10 M and B 1.1 d max pmol PTX3/pmol C1q (assuming for PTX3 the mass of the monomer, 45 kDa; mean of three independent experiments). Similar results were obtained also when nonbiotinylated puri- fied PTX3 was used. Results obtained using biotinylated PTX3 were confirmed in preliminary experiments where binding of unlabeled PTX3 to C1q was investigated by means of real time biomolecular interaction analysis with BIAcore®. As shown in Fig. 5, panel A, when C1q was immobilized on the sensor chip it was possible to observe a significant binding of PTX3, which was subsequently recognized by the specific antibody 1C8. Ki- netic analysis of the interaction between the two molecules 5 21 21 (Fig. 5, panel B) allowed to calculate a K of 2.4 3 10 M s on 24 21 and a K of 4 3 10 s . off The Role of the Pentraxin Domain—We wanted to obtain preliminary indications as to the role of the pentraxin domain of PTX3 in multimer formation and ligand recognition. A mu- tant consisting of the PTX3 pentraxin domain (starting from aa 179 of the mature protein, called short PTX3, sPTX3) was expressed in CHO cells. As shown in Fig. 6, panel A, sPTX3 migrated in SDS-PAGE under reducing conditions as two bands of 23 and 28 kDa. The 28-kDa band was drastically reduced by treatment with N-glycosidase F as described above for PTX3 (data not shown). Therefore, the two bands most likely represent unglycosylated and glycosylated sPTX3, con- sistent with the observation that the N-linked glycosylation FIG.2. Multimer formation by PTX3. Panel A shows the elution site is located within the pentraxin domain at aa 220 of the profile of PTX3 submitted to gel filtration on Superose 6 and eluted as detailed under “Experimental Procedures.” Arrows indicate molecular mature protein. Native sPTX3 did not form large multimers mass markers (ovalbumin, 43 kDa; catalase, 232 kDa; ferritin, 450 kDa; (decamers) and did not bind to C1q (Fig. 6, panel B). It is well thyroglobulin, 669 kDa; rabbit IgM, 900 kDa). Purified PTX3 was known that the classical short pentraxins CRP and SAP re- analyzed on denaturing (panels B and C) or native (panels D and E) gel. quired cross-linking for binding to C1q (13). As expected on this Panel B (Coomassie Blue-stained gel): lane 1, reduced PTX3; lane 2, reduced CRP; lane 3, unreduced PTX3; lane 4, unreduced CRP. Panel C basis, cross-linked sPTX3, consisting of multimers from >60- to (Western blot): lane 1, reduced PTX3; lane 2, unreduced PTX3. Panel D >220-kDa apparent molecular mass (data not shown), did bind (silver-stained gel): lane 1, unreduced PTX3; lane 2, unreduced SAP. C1q but not C1s. It is concluded that the recognition of C1q by Panel E: Western blot of unreduced native PTX3. PTX3 requires multimer formation and is mediated by the pentraxin domain of the molecule. Calcium—To investigate the possible presence of Ca DISCUSSION strongly coordinated to specific sites of the protein (as in SAP and CRP) inductive coupled plasma/atomic emission spectros- The present investigation was designed to express in mam- copy experiments were performed using protein samples puri- malian cells and to characterize the prototypic long pentraxin fied in Ca -free buffers. The emission profiles clearly show PTX3 and to compare its properties to those of classical pen- that the Ca content of the protein is comparable to that of a traxins. The first amino acid of human PTX3 expressed and control, indicating that PTX3 does not have a specific coordi- secreted by CHO cells is Glu of the cDNA-deduced sequence, nation site for Ca (data not shown). after removal of a signal peptide as predicted (27). The secreted Ligands—We analyzed the binding of PTX3 to the classical protein consists of a major 45-kDa form of the protomer, with a ligands recognized by CRP and SAP, namely PE, PC, and HPA minor 40-kDa component. Lectin binding and N-glycosidase (MObDG). As shown in Fig. 3, PTX3 does not appreciably bind treatment suggest that the 45-kDa form is glycosylated and any of these ligands, that, on the contrary, are recognized by that sugar moieties, presumably bound to the N-glycosylation CRP and/or SAP. site Asn (from now on residue numbering is based on mature A well known ligand for both CRP and SAP is the collagen- protein, without the leader peptide), account for 5 kDa. like C1q molecule, a component of the complement system. The A major objective of the present study was to test structural interaction of soluble PTX3 with immobilized C1q was ana- and functional predictions made in a previous investigation in lyzed, and a dose-dependent binding of PTX3 was observed, which PTX3 was modeled on the three-dimensional structure of which, on the contrary, did not react with bound C1s used as a SAP (7, 31). The pentraxin domain of PTX3 is accommodated control for nonspecific binding (Fig. 4, panel A). The data were comfortably in the three-dimensional scaffold of SAP, a conse- essentially similar when either the spent medium of CHO cells quence of the considerable degree of amino acid overall conser- transfected with the sense cDNA for PTX3 or the purified vation between the molecules (>50%). Nonetheless, several protein were assayed. Under these conditions PTX3 did not differences were highlighted, which could now be tested exper- bind type IV collagen, fibronectin, and gelatin (Fig. 4, panel B), imentally. In addition to the two Cys residues at position 193 while binding to H1 histone was observed (data not shown). and 254 (respectively 36 and 95 of SAP numbering) (31) con- Characterization of the Long Pentraxin PTX3 32821 FIG.3. Binding of PTX3 to PE, PC, or HPA. Panel A shows the binding of CRP and SAP to the classical pentraxin ligands immobilized to Sepharose. Data are expressed as milligrams/liter of the different proteins present in the bound or unbound fractions as evaluated by immu- noassay. Panel B shows the binding of PTX3 to the same ligands; data are ex- pressed as area under the curve after den- sitometric analysis of the exposed film, as detailed under “Experimental Procedures.” FIG.4. Binding of PTX3 to immobi- lized C1q, type IV collagen, fibronec- tin, and gelatin. Panel A shows the binding of supernatant from transfected and control cells and of purified PTX3. C1q was immobilized on polystyrene plates and incubated with PTX3 or Agg- IgG for 30 min at 37 °C. Binding was re- vealed by specific antibodies and enzyme- linked immunosorbent assay. Panel B, 100 ml of type IV collagen (Co IV,10 mg/ ml), fibronectin (FN) (10 mg/ml), or gela- tin (0.5%) were immobilized on plastic wells. Binding with biotinylated PTX3 (250 ng,2hat37 °C)was analyzed as detailed under “Experimental Proce- dures.” Panel C demonstrates the binding of C1q to immobilized PTX3. Different concentrations of purified PTX3 were im- mobilized on plastic plates and incubated with different amount of C1q for1hat 37 °C. S.E. for data of panel C was less than 0.05%. Panel D shows the specific binding of PTX3 to C1q. C1q was immo- bilized on plastic wells and incubated with different amounts of biotinylated PTX3. Specific binding was measured in accordance with a standard curve of bioti- nylated PTX3. served in all known members of the pentraxin family cloned so interchain disulfide bridge between some, but not all, subunits. far, PTX3 (human and mouse) shows four additional Cys in the Classical pentraxins are multimeric proteins composed of pentraxin domain and three in the non-pentraxin N-terminal variable numbers of subunits (1, 3). For instance human CRP is 162 340 portion. Cys and Cys were suggested to form an addi- a pentamer, as is generally the case for members of the family, tional intramolecular bridge, whereas the two tandems (posi- although SAP is composed of two pentameric disks interacting tions 30 and 32, 300 and 301) as well as Cys could engage in face-to-face and Limulus CRP is a hexamer (50, 54, 55). We inter- or intramolecular bonds (31). Multimers of human CRP found no evidence for pentamer formation by PTX3. Gel elec- and SAP are noncovalently linked and, as expected, denaturing trophoresis under nonreducing nondenaturing conditions conditions cause disaggregation and monomer formation (Fig. showed a major aggregate with an apparent molecular mass of 2, panel B) (7–9). Residues involved in multimer formation in approximately 440 kDa, suggesting that in this condition the CRP and SAP are not conserved in PTX3 (31). Accordingly, major PTX3 species is a decamer. Interestingly, comparison of denaturing with SDS did not disassemble the PTX3 440-kDa PTX3 with SAP revealed changes in all amino acids involved in multimers, and reduction with DTT was required for disaggre- interprotomer interactions (31). Based on this structural con- gation to the 45-kDa protomer. Similar results have been de- sideration, it was therefore not surprising to find that PTX3 scribed for apexin, another member of the long pentraxin group multimers differed considerably from the pentamer structure. (32, 33). Other classical pentraxins may have disulfide bridges On gel filtration, PTX3 eluted with an apparent molecular among the monomers, including plaice (46), dogfish (47, 48), mass of approximately 900 kDa, whereas on native gel electro- Xenopus (49), Limulus (50, 51), and rat CRP (52, 53). The latter phoresis the apparent size of the multimer was 440 kDa. Such is unique among the proteins of this family, since there is an a discrepancy is not without precedent for pentraxins and 32822 Characterization of the Long Pentraxin PTX3 FIG.6. Binding of the pentraxin domain of PTX3 to C1q. The pentraxin domain of PTX3, sPTX3, was analyzed on a reducing SDS- PAGE (A) and its binding to C1q was assessed (B). Panel A, Western blotting analysis: lane 1, 10-fold concentrated supernatant from sPTX3 transfected CHO cells; lane 2, 10-fold concentrated supernatant from antisense transfected CHO cells. Molecular mass markers are indicated on the left. Panel B, binding to C1q. PBS, C1q (500 ng/well), or C1s (500 ng/well) were immobilized on polystyrene plates and incubated with PBS, native sPTX3 (2 mg/well), or cross-linked sPTX3 (2 mg/well) for 30 min at 37 °C. Bound C1q was revealed as detailed under “Experimental Procedures.” PTX3 shares with CRP/SAP the ability to bind C1q, the first FIG.5. Specific interaction between C1q and PTX3 assessed component of the classical pathway of complement activation. with BIAcore®. Panel A, sensorgrams from a representative experi- The binding was specific in that other complement components ment are reported. Each pair of dots indicates the beginning and the (C1s) and other proteins, including collagen type IV which end of analyte injection, by which the increase in RU is calculated. binds SAP (57), were not recognized by PTX3. Using biotiny- Sensorgrams represent the interaction of immobilized C1q (base value 12,080 RU) with the anti-PTX3 antibody 1C8 (nonspecific binding of 54 lated PTX3, the estimated K was 7.4 3 10 M and a value in RU; identical to that obtained on the sensor chip without C1q, data not the same range was obtained when BIAcore® was used. B max shown) and with PTX3 (259 RU of binding). PTX3 bound to C1q was values indicate that one PTX3 protomer binds one C1q mole- subsequently recognized by the specific antibody 1C8 (285 RU of bind- cule. Using a similar methodological approach, a similar con- ing). Regeneration of the surface with NaOH was performed twice, after 1C8 injection. PanelB, saturation analysis of PTX3 binding to C1q. clusion was reached when the interaction of SAP with collagen Increasing concentrations of PTX3 (from 3 to 500 nM) were allowed to type IV was studied (57). interact with immobilized C1q for 6 min, and association curves were As predicted, recognition of C1q is mediated by the pentraxin recorded. Buffer was then run over and dissociation allowed to proceed. domain of PTX3. C1q binding by the pentraxin domain requires The kinetic parameters of the interaction were calculated for each 5 21 21 multimer formation, as classically observed for the short pen- sensogram. The mean values are K of 2.4 3 10 M s ; K of 4 3 on off 24 21 10 s . Data are from a single experiment, representative of five traxins CRP and SAP (13). It was predicted that Cys at position performed. 86 (mature protein) of the non-pentraxin portion may engage in interprotomer interactions (31). Artificial cross-linking of the isolated pentraxin domain of PTX3 (sPTX3), an absolute re- molecules such as collectins (56). It is tempting to speculate quirement for C1q recognition, may fulfill the same structural that PTX3 is predominantly assembled as a decamer, with function as interprotomer Cys bonds do in the native molecule. aggregates of two decamers being held by weak forces resolved Preliminary experiments indicate that PTX3 added to pooled upon electrophoresis. human serum causes the consumption of C4 and of the total SAP binds two Ca ions per monomer (7). The amino acid complement hemolytic activity as expected on the basis of C1q residues of SAP involved in the binding of Ca are not con- binding. If PTX3 recognizes microbial components, as sug- served in PTX3 (31). Inductive coupled plasma spectroscopy gested by preliminary data, in analogy with classical pentrax- experiments show that purified PTX3 does not bind calcium as ins, involvement of the complement system could regulate an- expected on the basis of structural analysis. Moreover, PTX3 timicrobial resistance, directly or indirectly via production of does not seem to bind to PC, PE, and HPA, all classical calcium- leukocyte chemotactic and activating fragments. dependent ligands of pentraxins (12, 17). PTX3 is the first cloned member of the long pentraxin family, Circular dichroism spectroscopy, performed on purified which includes XL-PXN1 from Xenopus (34), rat NP (35) and PTX3, showed that the protein is characterized by a predomi- the three homologue genes guinea pig apexin (32, 33), human nance of b-sheet secondary structure. Classical pentraxins are NTPX2 (36), and the latest rat neuronal pentraxin, Narp (37). characterized by a very high amount of b-sheet secondary No significant structural homologies are evident among the structure. Secondary structure prediction, performed on the different non-pentraxin domains, and dendogram analysis of N-terminal portion of PTX3 suggested a highly a-helical ar- the pentraxin domain suggests that human PTX3 and murine rangement for this domain (31). Even if the presence of some PTX3 may be as distantly related to long pentraxins as to a-helical component can be inferred from the CD spectral fea- tures of PTX3, the data suggest that the prediction overesti- mated the a-helical content of the protein. F. Tedesco and M. Pausa, unpublished results. Despite the structural and functional differences observed, B. Bottazzi and A. Bastone, unpublished results. Characterization of the Long Pentraxin PTX3 32823 16. Loveless, R. W., Floyd, O. S., Raynes, J. G., Yuen, C. T., and Feizi, T. (1992) classical pentraxins. It is interesting to observe that the long EMBO J. 11, 813– 819 pentraxins do not have the restricted liver inducibility typical 17. Hind, C. R., Collins, P. M., Renn, D., Cook, R. B., Caspi, D., Baltz, M. L., and of CRP and SAP (upon interleukin-6 stimulation) and show a Pepys, M. B. (1984) J. Exp. Med. 159, 1058 –1069 18. Volanakis, J. E. (1982) Ann. N. Y. Acad. Sci. 389, 235–250 more promiscuous pattern of expression in vitro and in vivo. 19. Ying, S. C., Gewurz, A. T., Jiang, H., and Gewurz, H. (1993) J. Immunol. 150, PTX3 can be expressed by endothelial cells, hepatocytes, fibro- 169 –176 blasts, and monocytes in response to lipopolysaccharide and 20. Jiang, H., Siegel, J. N., and Gewurz, H. (1991) J. Immunol. 146, 2324 –2330 21. Jiang, H., Robey, F. A., and Gewurz, H. (1992) J. Exp. Med. 175, 1373–1379 inflammatory cytokine (27, 29) and is induced by lipopolysac- 22. Bristow, C. L., and Boackle, R. J. (1986) Mol. Immunol. 23, 1045–1052 charide in vivo in heart and lung but not in liver (30, 31). 23. Pepys, M. B., Dyck, R. F., de Beer, F. C., Skinner, M., and Cohen, A. S. (1979) Clin. Exp. Immunol. 38, 284 –293 The results reported here show that the long pentraxin PTX3 24. de Beer, F. C., Baltz, M. L., Holford, S., Feinstein, A., and Pepys, M. B. (1981) exhibits structural and functional similarities as well as differ- J. Exp. Med. 154, 1134 –1139 ences when compared with the classical pentraxins CRP and 25. Garcia de Frutos, P., Hardig, Y., and Dahlback, B. (1995) J. Biol. Chem. 270, 26950 –26955 SAP. PTX3 forms multimers as CRP and SAP do, but these 26. Hamazaki, H. (1987) J. Biol. Chem. 262, 1456 –1460 differ in size and structural features (requirement for Cys 27. Breviario, F., d’Aniello, E. M., Golay, J., Peri, G., Bottazzi, B., Bairoch, A., bonds). PTX3 does not recognize the pentraxin ligands (Ca , Saccone, S., Marzella, R., Predazzi, V., Rocchi, M., Della Valle, G., Dejana, E., Mantovani, A., and Introna, M. (1992) J. Biol. Chem. 267, 22190 –22197 PE, PC, HPA) with the exception of C1q. This finding is con- 28. Lee, G. W., Lee, T. H., and Vilcek, J. (1993) J. Immunol. 150, 1804 –1812 sistent with the view that this pentraxin, secreted by macro- 29. Vidal Alles, V., Bottazzi, B., Peri, G., Golay, J., Introna, M., and Mantovani, A. (1994) Blood 84, 3483–3493 phages and endothelial cells following stimulation with inter- 30. Lee, G. W., Goodman, A. R., Lee, T. H., and Vilcek, J. (1994) J. Immunol. 153, leukin-1, tumor necrosis factor, and bacterial components, may 3700 –3707 contribute to the amplification of the effector mechanisms of 31. Introna, M., Vidal Alles, V., Castellano, M., Picardi, G., De Gioia, L., Bottazzi, B., Peri, G., Breviario, F., Salmona, M., De Gregorio, L., Dragani, T. A., innate immunity. In this regard, PTX3 seems to fulfill in tis- Srinivasan, N., Blundell, T. L., Hamilton, T. A., and Mantovani, A. (1996) sues the same function that liver-derived CRP and SAP exert Blood 87, 1862–1872 in the circulation. It remains to be elucidated whether and to 32. Reid, M. S., and Blobel, C. P. (1994) J. Biol. Chem. 269, 32615–32620 33. Noland, T. D., Friday, B. B., Maulit, M. T., and Gerton, G. L. (1994) J. Biol. what extent the observations reported herein for PTX3 can be Chem. 269, 32607–32614 extended to other recently identified long pentraxins. 34. Seery, L. T., Schoenberg, D. R., Barbaux, S., Sharp, P. M., and Whitehead, A. S. (1993) Proc. R. Soc. Lond. Ser. B Biol. Sci. 253, 263–270 Acknowledgments—We are grateful to Drs. Paul Proost and Jo Van 35. Schlimgen, A. K., Helms, J. A., Vogel, H., and Perin, M. S. (1995) Neuron 14, Damme for one of the two microsequence analyses of the purified 519 –526 protein and to Professor Mark B. Pepys for help with the binding assay 36. Hsu, Y. C., and Perin, M. S. (1995) Genomics 28, 220 –227 37. Tsui, C. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Barnes, C., and to Sepharose-immobilized ligands for measurement of CRP and SAP Worley, P. F. (1996) J. Neurosci. 15, 2463–2478 and for invaluable discussion. We also thank Dr. M. Gobbi for his help 38. Laemmli, U. K. (1970) Nature 227, 680 – 685 in the analysis of affinity and stoichiometry of PTX3 binding to C1q. 39. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197–203 REFERENCES 40. Taketa, K. (1987) Electrophoresis 8, 409 – 414 1. Osmand, A. P., Friedenson, B., Gewurz, H., Painter, R. H., Hofmann, T., and 41. Pepys, M. B., Dash, A. C., Markham, R. E., Thomas, H. C., Williams, B. D., and Shelton, E. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 739 –743 Petrie, A. (1978) Clin. Exp. Immunol. 32, 119 –124 2. Baltz, M. L., de Beer, F. C., Feinstein, A., Munn, E. A., Milstein, C. P., 42. Fiedel, B. A., Simpson, R. M., and Gewurz, H. 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Multimer Formation and Ligand Recognition by the Long Pentraxin PTX3

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 52, Issue of December 26, pp. 32817–32823, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 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* (Received for publication, August 6, 1997, and in revised form, October 6, 1997) Barbara Bottazzi‡§, Vale ´ rie Vouret-Craviari‡§, Antonio Bastone‡§, Luca De Gioia‡, Cristian Matteucci‡, Giuseppe Peri‡, Fabio Spreafico‡, Mario Pausa¶, Cinzia D’Ettorrei, Elisabetta Gianazza**, Aldo Tagliabuei, Mario Salmona‡, Francesco Tedesco‡‡, Martino Introna‡, and Alberto Mantovani‡§§¶¶ ‡From the Istituto di Ricerche Farmacologiche “Mario Negri,” Via Eritrea 62, 20157 Milano, Italy; ¶Istituto di Ostetricia e Ginecologia, IRCCS Burlo Garofolo, Trieste, Italy; iDOMPE’ S.P.A., L’Aquila, Italy; **Istituto di Scienze Farmacologiche, Universita ` di Milano, Milan, Italy; ‡‡Dipartimento di Fisiologia e Patologia, Universita ` di Trieste, Trieste, Italy; and §§Sezione di Patologia e Immunologia, Dipartimento di Biotecnologie, Universita ` di Brescia, Brescia, Italy PTX3 is a prototypic long pentraxin consisting of a quirement for disulfide bonds, and does not recognize C-terminal 203-amino acid pentraxin-like domain cou- CRP/SAP ligands. The capacity to bind C1q, mediated by pled with an N-terminal 178-amino acid unrelated por- the pentraxin domain, is consistent with the view that tion. The present study was designed to characterize the PTX3, produced in tissues by endothelial cells or macro- structure and ligand binding properties of human PTX3, phages in response to interleukin-1 and tumor necrosis in comparison with the classical pentraxins C-reactive factor, may act as a local regulator of innate immunity. protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 Pentraxins (C-reactive protein, CRP, and serum amyloid P (Glu). Lectin binding and treatment with N-glycosidase component, SAP) are acute phase proteins conserved during F showed that PTX3 is N-glycosylated, sugars account- evolution from Limulus polyphemus to man (1– 6). Pentraxins ing for 5 kDa of the monomer mass (45 kDa). Circular are composed of monomers with a b-jelly roll topology that dichroism analysis indicated that the protein consists usually assemble into pentameric, noncovalently associated b-sheets with a minor a-helical com- predominantly of structures (7–9). CRP and SAP are made in the liver in re- ponent. While in gel filtration the protein is eluted with sponse to inflammatory mediators, most prominently interleu- a molecular mass of >900 kDa, gel electrophoresis using kin-6 (10, 11). A number of ligands, recognized in a calcium- nondenaturing, nonreducing conditions revealed that dependent manner, have been identified for CRP and SAP, PTX3 forms multimers predominantly of 440 kDa appar- including phosphoethanolamine (PE), phosphocholine (PC) ent molecular mass, corresponding to decamers, and (12), DNA and chromatin (13–15), immune complexes, various that disulfide bonds are required for multimer forma- sugars (16), the best characterized of which is methyl 4,6-O-(1- tion. The ligand binding properties of PTX3 were then carboxyethylidene)-b-D-galactopyranoside (MObDG) (17), and examined. As predicted based on modeling, inductive complement components (13, 18 –22). Moreover, SAP binds to coupled plasma/atomic emission spectroscopy showed all forms of amyloid fibrils (23) and, in addition, to fibronectin, . Unlike the that PTX3 does not have coordinated Ca C4-binding protein (24, 25), and glycosaminoglycans (26). Pen- classical pentraxins CRP and SAP, PTX3 did not bind traxins represent a mechanism of innate resistance against phosphoethanolamine, phosphocholine, or high pyru- vate agarose. PTX3 in solution, bound to immobilized microbes, tools to scavenge cellular debris and components of C1q, but not C1s, and, reciprocally, C1q bound to immo- the extracellular matrix as illustrated by amyloid deposits (3). bilized PTX3. Binding of PTX3 to C1q is specific and PTX3 is a prototypic long pentraxin, structurally related to, 7.4 3 10 M as determined by solid saturable with a K d yet distinct from, classical pentraxins. PTX3 was cloned as an phase binding assay. The Chinese hamster ovary cell- interleukin-1-inducible gene in endothelial cells (27) and as a expressed pentraxin domain bound C1q when multim- tumor necrosis factor-inducible gene (TSG 14) in fibroblasts erized. Thus, as predicted on the basis of computer mod- (28). PTX3 is constituted of a C-terminal pentraxin-like domain eling, the prototypic long pentraxin PTX3 forms of 203 amino acids encoded by the third exon and by an N- multimers, which differ from those formed by classical terminal 178- amino acid long portion (27). Inflammatory cy- pentraxins in terms of protomer composition and re- tokines induce PTX3 expression in a variety of cell types, most prominently endothelial cells and mononuclear phagocytes (27, 29 –31). After cloning of PTX3, other “long pentraxins” were * This work was supported by the Associazione Italiana Ricerca sul identified, including guinea pig apexin (32, 33), XL-PXN1 from Cancro (AIRC), by the special project Biotechnology, Consiglio Nazion- ale delle Ricerche (CNR), by Istituto Superiore di Sanita ` (project On- cology), and by Ministero della Ricerca Scientifica e Tecnologica (40 and 60%), Italy. The costs of publication of this article were defrayed in part The abbreviations used are: CRP, C-reactive protein; SAP, serum by the payment of page charges. This article must therefore be hereby amyloid P component; PE, phosphoethanolamine; PC, phosphocholine; marked “advertisement” in accordance with 18 U.S.C. Section 1734 MObDG, methyl 4,6-O-(1-carboxyethylidene)-b-D-galactopyranoside; solely to indicate this fact. PBS, phosphate-buffered saline; HPA, high pyruvate agarose; NP, neu- § These authors have contributed equally to this work. ronal pentraxin; bp, base pair(s); CHO, Chinese hamster ovary; PAGE, ¶¶ To whom correspondence should be addressed: Fax: 39/2/3546277; polyacrylamide gel electrophoresis; DTT, dithiotreitol; RU, resonance E-mail: [email protected]. units. This paper is available on line at http://www.jbc.org 32817 This is an Open Access article under the CC BY license. 32818 Characterization of the Long Pentraxin PTX3 pH 7.4, 1% Triton X-100, 0.1% SDS, and 1 unit of N-glycosidase F Xenopus (34), rat neuronal pentraxin (NP) (35), human neuro- (Boehringer Mannheim GmbH, Mannheim, Germany). After overnight nal pentraxin (NPTX2) (36), which possibly represents the incubation at room temperature, samples were analyzed by SDS-PAGE. human homologue of apexin, and Narp (37), which possibly Purification and Cross-linking of sPTX3—Supernatant from sPTX3 represents the rat homologue of apexin. In all these molecules cells was concentrated by ultrafiltration, and the buffer was changed to a C-terminal pentraxin domain is coupled to diverse, unrelated 50 mM Tris-HCl, pH 7, before application on a HR 5/5 Mono Q column. N-terminal portions (32–36). The structure and function of long sPTX3 was eluted with a linear gradient of NaCl (from 0 to 1 M)in50 mM Tris-HCl. Fractions recognized by the 16B5 monoclonal antibody pentraxins is unknown. were pooled, concentrated by ultrafiltration, and subjected to cross- The present study was designed to express the prototypic linking. sPTX3 (200 mgin200 ml of Veronal-buffered saline) was cross- long pentraxin PTX3 in an eukaryotic system and to charac- linked by adding 20 mlof10mM bis(sulfosuccinimidyl)suberate (Pierce) terize its structure and ligand recognition in comparison to for1hat room temperature. Then 40 ml of Tris-buffered saline was classical pentraxins. In particular, experiments were designed added to stop the reaction. to test predictions made on the basis of modeling of the pen- Ligand Binding Assays—Binding of PTX3 with Sepharose-immobi- lized PE, Sepharose-immobilized PC, or high piruvate agarose (HPA) traxin domain of PTX3 on the three-dimensional structure of was performed as described previously for CRP and SAP (41). The SAP (31). presence of PTX3 was assayed by Western blot, and results are ex- pressed as area under the curve after densitometric analysis of the EXPERIMENTAL PROCEDURES exposed film performed by the scanning densitometer GS300 (Hoefer Production of Recombinant PTX3—A 1311-bp fragment of human Scientific Instrument, San Francisco, CA). As a control, acute phase PTX3 cDNA, containing the complete coding sequence, was subcloned human serum was incubated in parallel with immobilized ligand and in pSG5 vector (Stratagene, La Jolla, CA) and transfected in CHO cells processed as for PTX3, then assayed for the presence of CRP and/or SAP by calcium phosphate precipitation. Two clones selected with G418 (Life by electroimmunoassay as described previously (41). Technologies, Inc., Paisley, Scotland, UK) were used in the present Binding of PTX3 to C1q was performed as described previously for study, CHO 3.5, producing high levels of PTX3, and CHO 2.1, trans- CRP and SAP (19, 20). Briefly 96-well plates were coated with 250 –500 fected with the antisense construct. Conditioned medium was collected ng of C1q (Calbiochem) in PBS with calcium and magnesium (4 °C from confluent monolayers incubated 24 h with culture medium (Dul- overnight). Wells were washed with PBS plus 0.05% Tween 20, blocked becco’s modified Eagle’s medium; Seromed, Berlin, Germany) without with 0.5% dry milk in PBS (2 h at room temperature), and extensively fetal calf serum. The pentraxin domain of PTX3 was obtained by intro- washed before the addition of 100 ml of supernatant from 3.5 or 2.1 cell duction by polymerase chain reaction of a XhoI site at the end of the lines or 200 ng of purified PTX3 diluted in PBS (30 min, 37 °C). After signal peptide (position 18 of the published sequence) (27) and at the 59 washing, plates were incubated with 16B5 monoclonal antibody (1 h at end of the pentraxin domain (position 172). The fragment was sub- room temperature), washed again, and incubated with horseradish cloned in pSG5 vector and transfected in CHO cells by calcium phos- peroxidase-labeled goat anti-rat IgG (Amersham; 1:2000;1hat room phate precipitation as described above. A high recombinant producer temperature). After extensive washing, 100 ml of chromogen substrate clone, named sPTX3, was selected. ABTS were added (Kirkegaard and Perry, Gaithersburg, MD), and Gel Electrophoresis and Western Blot Analysis—5–10% polyacryl- absorbance values were read at 405 nm. As positive control for the amide gradient gel in the presence of SDS was run in the discontinuous binding assay, immobilized rabbit IgG heated at 63 °C for 20 min buffer system of Laemmli (38). Gels were stained with Amido Black, immediately prior to use was used (Agg-IgG) (42). Coomassie Brilliant Blue, or silver nitrate (39). For Western blots, In another set of experiments, wells were coated by overnight incu- separated proteins were electroblotted onto nitrocellulose filters (Hy- bation at 4 °C with 200 ml of purified PTX3 (2.5–10 mg/ml) in 100 mM bond ECL, Amersham Corp.) and labeled with anti-PTX3 monoclonal sodium carbonate, pH 9.6. After blocking, C1q was added in amounts antibody (see below) following standard procedures. Labeled proteins varying between 0.125 and 2 mg/ml, and incubation was continued for were detected by enhanced chemiluminescence (ECL, Amersham Corp.) 1 h at 37 °C. The bound C1q was revealed by its reaction with a specific in accordance with the manufacturer’s instructions. PTX3 was analyzed rabbit antibody for1hat37 °C followed, after washing, by biotin- in the native state in 5–10% gradient polyacrylamide gel electrophore- labeled goat anti-rabbit IgG for an additional h at 37 °C. The wells were sis (PAGE). washed and incubated with 200 ml of alkaline phosphatase-conjugated Protein Purification—Culture supernatant from CHO 3.5 cells was streptavidin (Jackson Laboratories, West Grove, PA) diluted 1/8000 for concentrated, and the buffer was changed to 50 mM imidazole, pH 6.6, 30 min at 37 °C. The enzymatic reaction was developed using the before application on a HR 5/5 Mono Q column (Pharmacia Biotech, substrate p-nitrophenyl phosphate (Sigma; 1 mg/ml) in 0.1 M glycin Uppsala, Sweden) preequilibrated with the same buffer. The column buffer pH 10.4 containing 0.1 mM MgCl and 0.1 mM ZnCl . In some 2 2 was washed until the absorbance was stable, and PTX3 was eluted with experiments C1q purified from human plasma following the procedure 1 M NaCl in 50 mM imidazole at 1 ml/min using a nonlinear gradient. In originally described by Tenner et al. (43) was used with similar results. the first step, the NaCl concentration was increased from 0% to 58% in Binding of PTX3 to C1q was characterized using biotin-labeled pu- 35 min. Then the NaCl concentration was immediately increased to rified PTX3 (bPTX3). Concentrations ranging from 25 to 800 ng of 100%, and PTX3 was obtained as a narrow peak as monitored by UV bPTX3 (0.56 –17.92 pmol considering a molecular mass of 45 kDa for detection at 280 nm. The PTX3-containing fraction was subjected to gel PTX3 monomer) in 100 ml were added to triplicate wells coated with 200 filtration on Sephacryl S-300, and PTX3 was finally eluted with PBS. ng of C1q (0.49 pmol). After 1 h incubation at 37 °C, bound PTX3 was The purification of PTX3 was monitored by SDS-PAGE and Western detected with horseradish peroxidase-labeled avidin (1/2000, 1 h) and blot analysis. To further control the elution profile, purified PTX3 was chromogen substrate ABTS, as described. The results were converted to applied to a Superose 6 column (Pharmacia Biotech) calibrated with picomolar concentration using a standard curve of bPTX3 and consid- molecular weight standards and eluted with PBS at a flow rate of 0.4 ering a molecular mass of 45 kDa for PTX3 monomer. K and B were d max ml/min. Elution was monitored by UV detection at 280 nm, and frac- obtained by nonlinear fitting of the saturation curves (44). Binding of tions (2 min each) were subsequently analyzed on native PAGE. bPTX3 to type IV collagen (Sigma; 10 mg/ml), fibronectin (Sigma; 10 Antibodies—A rat (16B5) and mouse (1C8) antibody against human mg/ml), and gelatin (Sigma; 0.5%) was analyzed with essentially the PTX3 were used in this study. Rabbit antiserum to human C1q was same protocol. purchased from Istituto Behring SpA (Scoppito, Italy), and biotin-la- Biosensor Analysis—The interaction of PTX3 with C1q was also beled goat anti-rabbit IgG was obtained from Sigma. analyzed with the BIAcore® system (biomolecular interaction analysis; Lectin Staining and Deglycosylation—Purified PTX3 (5 mg) after BIAcore AB, Uppsala, Sweden). C1q was immobilized on a CM5 sensor SDS-PAGE and electroblotting was incubated with the following lectin- chip (Pharmacia Biosensor) using the amine coupling kit (Pharmacia peroxidase conjugates at a concentration of 50 mg/ml: concanavalin A, Biosensor) (45). A volume of 30 ml of ligand (10 mg/ml C1q in 10 mM wheat germ agglutinin, Bandeiraea simplicifolia lectin BS-II, Vicia sodium acetate, pH 4.1) was immobilized with a continuous flow of HBS faba lectin, Psophocarpus tetragonolobus lectin, and Tetragonolobus (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% BIAcore® purpureas lectin. The zymogram for peroxidase was developed accord- surfactant P20, pH 7.4) at 5 ml/min. Each binding assay was performed ing to Taketa (40) in a solution containing 2 mg/ml NADH, 0.6 mg/ml with a constant flow rate (5 ml/min) of HBS, pH 7.4 at 25 °C. The nitro blue tetrazolium, 0.4 mg/ml phenol, and 3 ml/ml H O , in phos- analyte PTX3 was injected over the ligand surface in HBS and the 2 2 phate buffer, pH 7.0. Purified PTX3 (160 mg in PBS) was first made 1% surface was then regenerated by injection of 5 mlof25mM NaOH. in SDS and incubated at 100 °C for 5 min, then diluted 5-fold with Analyte binding was calculated as the difference in RU before and after concentrated buffer to a final concentration of 50 mM sodium phosphate, the interaction of ligand with analyte. For saturation analysis, PTX3 Characterization of the Long Pentraxin PTX3 32819 siderable donor to donor variation (29). To test for the pres- ence of oligosaccharides, PTX3 was stained with different lectin-peroxidase conjugates. PTX3 showed a strong staining for peroxidase-conjugated concanavalin A (specific for a-D-man- nose and a-D-glucose) and wheat germ agglutinin (specific for (D-GlcNAc)2NeuAc, Fig. 1, panel B) while it was not stained by B. simplicifolia, V. faba, P. tetragonolobus, and T. purpureas (data not shown). To better characterize the glycosylation of PTX3, the purified protein was treated with N-glycosidase F, an enzyme that cleaves Asn-linked high mannose as well as hybrid and com- plex oligosaccarides, and the deglycosylation was monitored as FIG.1. Purification and glycosylation of PTX3 expressed in CHO cells. Panel A, proteins were separated on 4 –10% SDS-PAGE and an increase in electrophoretic mobility. As shown in Fig. 1, analyzed by Western blotting (lanes 1 and 2) and silver staining (lane panel C, after treatment with N-glycosidase F, PTX3 exhibited 3). Lane 1, supernatant from CHO 3.5 cells; lanes 2 and 3, the same a decrease of approximately 5 kDa, from 45 to 40 kDa. It is after purification. Panel B, purified protein was run on 7.5–17.5% concluded that PTX3 is glycosylated and that N-linked sugars SDS-PAGE and stained with Amido Black (lane 1), concanavalin A (lane 2), or wheat germ agglutinin (lane 3). Panel C, effect of enzymatic account for 5 kDa of the major 45-kDa PTX3 protomer. Isoelec- deglycosylation on PTX3. Purified PTX3 was denatured by SDS and trofocusing of purified PTX3 showed five different bands with a incubated in the absence (lane 1) or presence of N-glycosidase F (lane 2) pI ranging from 4.5 to 4.65 (data not shown), possibly indicat- overnight at room temperature. Control and enzyme-treated samples ing heterogeneity in glycosylation in agreement with a previous were analyzed by Western blot after SDS-PAGE. The molecular mass standards (3 10 ) are indicated on the left. suggestion (29). Conformation and Multimer Formation—The purified mate- rial was used to generate a CD spectrum to determine the was serially diluted in HBS to concentrations ranging from 20 to 600 possible conformation of the protein. The spectrum is charac- nM. Each sample (30 ml) was injected and allowed a total contact time terized by a positive band at 199 nm and a negative band at 217 with the ligand surface of 6 min. nm. Careful observation of the negative band shows the pres- ence of shoulders at 209 and 224 nm. Spectral deconvolution RESULTS indicates a predominantly b-sheet protein, with some contri- Production and Purification of Human Recombinant PTX3— bution of a-helical structure (data not shown). The culture supernatant of the CHO 3.5 cell line was analyzed Classical pentraxins form noncovalently linked multimers by SDS-PAGE under reducing conditions, and a protein with (7–9). In an effort to obtain indications as to the capacity of an apparent molecular mass of 45 kDa, which was not present PTX3 to form multimers, purified protein was analyzed by gel in the culture supernatant from the antisense clone 2.1 (not filtration on Superose 6. As shown in Fig. 2, panel A,ongel shown), was observed. Western blot analysis of the same cul- filtration PTX3 eluted with an apparent molecular mass of ture supernatant showed that this protein is recognized by the about 900 kDa (similar results were obtained also with monoclonal anti-PTX3 antibody 16B5 (Fig. 1, panel A, lane 1). Sephacryl S300). When fractions from gel filtration containing To purify the protein, 500 ml of culture supernatant from the protein were run on a native gel, a pattern similar to that CHO 3.5 cells were collected, concentrated by ultrafiltration, shown in Fig. 2, panels D and E (see below) was observed. and then subjected to ion exchange chromatography on a Mono To further characterize the PTX3 multimers, denaturation with SDS and reduction with DTT were used. The apparent Q column. The fractions containing PTX3 were subsequently size of the PTX3 multimers was determined by gel electro- subjected to gel filtration in PBS. The process of purification phoresis followed by staining or Western blotting. In the ab- was monitored by SDS-PAGE and by microsequence analysis sence of reducing agents, SDS treatment caused disassembly of (see below). The purification scheme yielded PTX3 prepara- the multimers formed by SAP (not shown) and CRP (Fig. 2, tions of considerable purity (Fig. 1, panel A, lanes 2 and 3) with panel B), to their constituent monomers. In contrast, under the an occasional 66-kDa contaminant. About 5 mg of pure PTX3 same conditions, SDS treatment in the absence of DTT did not were routinely obtained from 500 ml of conditioned medium. disaggregate PTX3, which migrated as two high molecular Microsequence analysis, performed on two occasions on the N mass bands that barely entered the gel (Fig. 2, panels B and C). terminus of the purified protein, confirmed the purity and SAP multimers are clearly visible under nondenaturing, non- showed identity with the amino acid sequence predicted on the reducing conditions as a major 230-kDa band (Fig. 2, panel D). basis of the cDNA (Glu-Asn-Ser-Asp-Asp-Tyr-Asp-Leu-Met- Under the same native conditions PTX3 migrates in the gel as Tyr-Val-Asn-Leu-Asp-Asn-Glu-Ile); it demonstrates that the a predominant 440-kDa apparent molecular mass species, pos- predicted leader peptide is removed when the protein is se- sibly corresponding to a decamer, as revealed by direct staining creted, the first sequenced amino acid being Glu of the pub- and Western blotting (Fig. 2, panels D and E). In addition, two lished sequence (27, 31). minor higher forms in the 540 – 600 kDa range were usually Glycosylation—The amino acid sequence predicts a molecu- visible. This relative distribution of different multimeric forms lar mass for the reduced protein of 40 kDa instead of the 45 was observed both in silver-stained gel and in Western blot kDa observed in SDS-PAGE. The PTX3 sequence shows the (Fig. 2, panel D and E) and is not dependent on concentration, presence of a potential N-linked glycosylation site at amino since gel filtration and PAGE performed with diluted PTX3 (up acid position 220 (27). SDS-PAGE of the reduced protein shows to the sensitivity of these assays) gave similar results (not the presence of two closely related bands (Fig. 1, panels A and shown). When PTX3 was incubated with increasing concentra- C). The lower band has a calculated molecular mass of 40 kDa, tions of DTT prior to SDS-PAGE, progressively smaller aggre- the size expected for PTX3 on the basis of the amino acidic gates were observed until, at 1 mM DTT, all protein ran in the sequence without the leader peptide. The amount of 40-kDa form of the 45-kDa protomer (not shown). material was variable from preparation to preparation. It is noteworthy that the same two bands were present in superna- tants of stimulated endothelial cells and monocytes, with con- N. Polentarutti and M. Introna, unpublished results. 32820 Characterization of the Long Pentraxin PTX3 Binding of PTX3 to C1q was also observed when soluble C1q was tested on immobilized PTX3 (Fig. 4, panel C). To characterize the binding of PTX3 to C1q, serial dilutions of biotinylated PTX3 were added to immobilized C1q and the amount of bound PTX3 evaluated on the basis of a standard curve of the biotinylated protein. Fig. 4, panel D, represents a typical experiment showing the binding of PTX3 to C1q; the binding is saturable with a K of 7.4 3 10 M and B 1.1 d max pmol PTX3/pmol C1q (assuming for PTX3 the mass of the monomer, 45 kDa; mean of three independent experiments). Similar results were obtained also when nonbiotinylated puri- fied PTX3 was used. Results obtained using biotinylated PTX3 were confirmed in preliminary experiments where binding of unlabeled PTX3 to C1q was investigated by means of real time biomolecular interaction analysis with BIAcore®. As shown in Fig. 5, panel A, when C1q was immobilized on the sensor chip it was possible to observe a significant binding of PTX3, which was subsequently recognized by the specific antibody 1C8. Ki- netic analysis of the interaction between the two molecules 5 21 21 (Fig. 5, panel B) allowed to calculate a K of 2.4 3 10 M s on 24 21 and a K of 4 3 10 s . off The Role of the Pentraxin Domain—We wanted to obtain preliminary indications as to the role of the pentraxin domain of PTX3 in multimer formation and ligand recognition. A mu- tant consisting of the PTX3 pentraxin domain (starting from aa 179 of the mature protein, called short PTX3, sPTX3) was expressed in CHO cells. As shown in Fig. 6, panel A, sPTX3 migrated in SDS-PAGE under reducing conditions as two bands of 23 and 28 kDa. The 28-kDa band was drastically reduced by treatment with N-glycosidase F as described above for PTX3 (data not shown). Therefore, the two bands most likely represent unglycosylated and glycosylated sPTX3, con- sistent with the observation that the N-linked glycosylation FIG.2. Multimer formation by PTX3. Panel A shows the elution site is located within the pentraxin domain at aa 220 of the profile of PTX3 submitted to gel filtration on Superose 6 and eluted as detailed under “Experimental Procedures.” Arrows indicate molecular mature protein. Native sPTX3 did not form large multimers mass markers (ovalbumin, 43 kDa; catalase, 232 kDa; ferritin, 450 kDa; (decamers) and did not bind to C1q (Fig. 6, panel B). It is well thyroglobulin, 669 kDa; rabbit IgM, 900 kDa). Purified PTX3 was known that the classical short pentraxins CRP and SAP re- analyzed on denaturing (panels B and C) or native (panels D and E) gel. quired cross-linking for binding to C1q (13). As expected on this Panel B (Coomassie Blue-stained gel): lane 1, reduced PTX3; lane 2, reduced CRP; lane 3, unreduced PTX3; lane 4, unreduced CRP. Panel C basis, cross-linked sPTX3, consisting of multimers from >60- to (Western blot): lane 1, reduced PTX3; lane 2, unreduced PTX3. Panel D >220-kDa apparent molecular mass (data not shown), did bind (silver-stained gel): lane 1, unreduced PTX3; lane 2, unreduced SAP. C1q but not C1s. It is concluded that the recognition of C1q by Panel E: Western blot of unreduced native PTX3. PTX3 requires multimer formation and is mediated by the pentraxin domain of the molecule. Calcium—To investigate the possible presence of Ca DISCUSSION strongly coordinated to specific sites of the protein (as in SAP and CRP) inductive coupled plasma/atomic emission spectros- The present investigation was designed to express in mam- copy experiments were performed using protein samples puri- malian cells and to characterize the prototypic long pentraxin fied in Ca -free buffers. The emission profiles clearly show PTX3 and to compare its properties to those of classical pen- that the Ca content of the protein is comparable to that of a traxins. The first amino acid of human PTX3 expressed and control, indicating that PTX3 does not have a specific coordi- secreted by CHO cells is Glu of the cDNA-deduced sequence, nation site for Ca (data not shown). after removal of a signal peptide as predicted (27). The secreted Ligands—We analyzed the binding of PTX3 to the classical protein consists of a major 45-kDa form of the protomer, with a ligands recognized by CRP and SAP, namely PE, PC, and HPA minor 40-kDa component. Lectin binding and N-glycosidase (MObDG). As shown in Fig. 3, PTX3 does not appreciably bind treatment suggest that the 45-kDa form is glycosylated and any of these ligands, that, on the contrary, are recognized by that sugar moieties, presumably bound to the N-glycosylation CRP and/or SAP. site Asn (from now on residue numbering is based on mature A well known ligand for both CRP and SAP is the collagen- protein, without the leader peptide), account for 5 kDa. like C1q molecule, a component of the complement system. The A major objective of the present study was to test structural interaction of soluble PTX3 with immobilized C1q was ana- and functional predictions made in a previous investigation in lyzed, and a dose-dependent binding of PTX3 was observed, which PTX3 was modeled on the three-dimensional structure of which, on the contrary, did not react with bound C1s used as a SAP (7, 31). The pentraxin domain of PTX3 is accommodated control for nonspecific binding (Fig. 4, panel A). The data were comfortably in the three-dimensional scaffold of SAP, a conse- essentially similar when either the spent medium of CHO cells quence of the considerable degree of amino acid overall conser- transfected with the sense cDNA for PTX3 or the purified vation between the molecules (>50%). Nonetheless, several protein were assayed. Under these conditions PTX3 did not differences were highlighted, which could now be tested exper- bind type IV collagen, fibronectin, and gelatin (Fig. 4, panel B), imentally. In addition to the two Cys residues at position 193 while binding to H1 histone was observed (data not shown). and 254 (respectively 36 and 95 of SAP numbering) (31) con- Characterization of the Long Pentraxin PTX3 32821 FIG.3. Binding of PTX3 to PE, PC, or HPA. Panel A shows the binding of CRP and SAP to the classical pentraxin ligands immobilized to Sepharose. Data are expressed as milligrams/liter of the different proteins present in the bound or unbound fractions as evaluated by immu- noassay. Panel B shows the binding of PTX3 to the same ligands; data are ex- pressed as area under the curve after den- sitometric analysis of the exposed film, as detailed under “Experimental Procedures.” FIG.4. Binding of PTX3 to immobi- lized C1q, type IV collagen, fibronec- tin, and gelatin. Panel A shows the binding of supernatant from transfected and control cells and of purified PTX3. C1q was immobilized on polystyrene plates and incubated with PTX3 or Agg- IgG for 30 min at 37 °C. Binding was re- vealed by specific antibodies and enzyme- linked immunosorbent assay. Panel B, 100 ml of type IV collagen (Co IV,10 mg/ ml), fibronectin (FN) (10 mg/ml), or gela- tin (0.5%) were immobilized on plastic wells. Binding with biotinylated PTX3 (250 ng,2hat37 °C)was analyzed as detailed under “Experimental Proce- dures.” Panel C demonstrates the binding of C1q to immobilized PTX3. Different concentrations of purified PTX3 were im- mobilized on plastic plates and incubated with different amount of C1q for1hat 37 °C. S.E. for data of panel C was less than 0.05%. Panel D shows the specific binding of PTX3 to C1q. C1q was immo- bilized on plastic wells and incubated with different amounts of biotinylated PTX3. Specific binding was measured in accordance with a standard curve of bioti- nylated PTX3. served in all known members of the pentraxin family cloned so interchain disulfide bridge between some, but not all, subunits. far, PTX3 (human and mouse) shows four additional Cys in the Classical pentraxins are multimeric proteins composed of pentraxin domain and three in the non-pentraxin N-terminal variable numbers of subunits (1, 3). For instance human CRP is 162 340 portion. Cys and Cys were suggested to form an addi- a pentamer, as is generally the case for members of the family, tional intramolecular bridge, whereas the two tandems (posi- although SAP is composed of two pentameric disks interacting tions 30 and 32, 300 and 301) as well as Cys could engage in face-to-face and Limulus CRP is a hexamer (50, 54, 55). We inter- or intramolecular bonds (31). Multimers of human CRP found no evidence for pentamer formation by PTX3. Gel elec- and SAP are noncovalently linked and, as expected, denaturing trophoresis under nonreducing nondenaturing conditions conditions cause disaggregation and monomer formation (Fig. showed a major aggregate with an apparent molecular mass of 2, panel B) (7–9). Residues involved in multimer formation in approximately 440 kDa, suggesting that in this condition the CRP and SAP are not conserved in PTX3 (31). Accordingly, major PTX3 species is a decamer. Interestingly, comparison of denaturing with SDS did not disassemble the PTX3 440-kDa PTX3 with SAP revealed changes in all amino acids involved in multimers, and reduction with DTT was required for disaggre- interprotomer interactions (31). Based on this structural con- gation to the 45-kDa protomer. Similar results have been de- sideration, it was therefore not surprising to find that PTX3 scribed for apexin, another member of the long pentraxin group multimers differed considerably from the pentamer structure. (32, 33). Other classical pentraxins may have disulfide bridges On gel filtration, PTX3 eluted with an apparent molecular among the monomers, including plaice (46), dogfish (47, 48), mass of approximately 900 kDa, whereas on native gel electro- Xenopus (49), Limulus (50, 51), and rat CRP (52, 53). The latter phoresis the apparent size of the multimer was 440 kDa. Such is unique among the proteins of this family, since there is an a discrepancy is not without precedent for pentraxins and 32822 Characterization of the Long Pentraxin PTX3 FIG.6. Binding of the pentraxin domain of PTX3 to C1q. The pentraxin domain of PTX3, sPTX3, was analyzed on a reducing SDS- PAGE (A) and its binding to C1q was assessed (B). Panel A, Western blotting analysis: lane 1, 10-fold concentrated supernatant from sPTX3 transfected CHO cells; lane 2, 10-fold concentrated supernatant from antisense transfected CHO cells. Molecular mass markers are indicated on the left. Panel B, binding to C1q. PBS, C1q (500 ng/well), or C1s (500 ng/well) were immobilized on polystyrene plates and incubated with PBS, native sPTX3 (2 mg/well), or cross-linked sPTX3 (2 mg/well) for 30 min at 37 °C. Bound C1q was revealed as detailed under “Experimental Procedures.” PTX3 shares with CRP/SAP the ability to bind C1q, the first FIG.5. Specific interaction between C1q and PTX3 assessed component of the classical pathway of complement activation. with BIAcore®. Panel A, sensorgrams from a representative experi- The binding was specific in that other complement components ment are reported. Each pair of dots indicates the beginning and the (C1s) and other proteins, including collagen type IV which end of analyte injection, by which the increase in RU is calculated. binds SAP (57), were not recognized by PTX3. Using biotiny- Sensorgrams represent the interaction of immobilized C1q (base value 12,080 RU) with the anti-PTX3 antibody 1C8 (nonspecific binding of 54 lated PTX3, the estimated K was 7.4 3 10 M and a value in RU; identical to that obtained on the sensor chip without C1q, data not the same range was obtained when BIAcore® was used. B max shown) and with PTX3 (259 RU of binding). PTX3 bound to C1q was values indicate that one PTX3 protomer binds one C1q mole- subsequently recognized by the specific antibody 1C8 (285 RU of bind- cule. Using a similar methodological approach, a similar con- ing). Regeneration of the surface with NaOH was performed twice, after 1C8 injection. PanelB, saturation analysis of PTX3 binding to C1q. clusion was reached when the interaction of SAP with collagen Increasing concentrations of PTX3 (from 3 to 500 nM) were allowed to type IV was studied (57). interact with immobilized C1q for 6 min, and association curves were As predicted, recognition of C1q is mediated by the pentraxin recorded. Buffer was then run over and dissociation allowed to proceed. domain of PTX3. C1q binding by the pentraxin domain requires The kinetic parameters of the interaction were calculated for each 5 21 21 multimer formation, as classically observed for the short pen- sensogram. The mean values are K of 2.4 3 10 M s ; K of 4 3 on off 24 21 10 s . Data are from a single experiment, representative of five traxins CRP and SAP (13). It was predicted that Cys at position performed. 86 (mature protein) of the non-pentraxin portion may engage in interprotomer interactions (31). Artificial cross-linking of the isolated pentraxin domain of PTX3 (sPTX3), an absolute re- molecules such as collectins (56). It is tempting to speculate quirement for C1q recognition, may fulfill the same structural that PTX3 is predominantly assembled as a decamer, with function as interprotomer Cys bonds do in the native molecule. aggregates of two decamers being held by weak forces resolved Preliminary experiments indicate that PTX3 added to pooled upon electrophoresis. human serum causes the consumption of C4 and of the total SAP binds two Ca ions per monomer (7). The amino acid complement hemolytic activity as expected on the basis of C1q residues of SAP involved in the binding of Ca are not con- binding. If PTX3 recognizes microbial components, as sug- served in PTX3 (31). Inductive coupled plasma spectroscopy gested by preliminary data, in analogy with classical pentrax- experiments show that purified PTX3 does not bind calcium as ins, involvement of the complement system could regulate an- expected on the basis of structural analysis. Moreover, PTX3 timicrobial resistance, directly or indirectly via production of does not seem to bind to PC, PE, and HPA, all classical calcium- leukocyte chemotactic and activating fragments. dependent ligands of pentraxins (12, 17). PTX3 is the first cloned member of the long pentraxin family, Circular dichroism spectroscopy, performed on purified which includes XL-PXN1 from Xenopus (34), rat NP (35) and PTX3, showed that the protein is characterized by a predomi- the three homologue genes guinea pig apexin (32, 33), human nance of b-sheet secondary structure. Classical pentraxins are NTPX2 (36), and the latest rat neuronal pentraxin, Narp (37). characterized by a very high amount of b-sheet secondary No significant structural homologies are evident among the structure. Secondary structure prediction, performed on the different non-pentraxin domains, and dendogram analysis of N-terminal portion of PTX3 suggested a highly a-helical ar- the pentraxin domain suggests that human PTX3 and murine rangement for this domain (31). Even if the presence of some PTX3 may be as distantly related to long pentraxins as to a-helical component can be inferred from the CD spectral fea- tures of PTX3, the data suggest that the prediction overesti- mated the a-helical content of the protein. F. Tedesco and M. Pausa, unpublished results. Despite the structural and functional differences observed, B. Bottazzi and A. Bastone, unpublished results. Characterization of the Long Pentraxin PTX3 32823 16. Loveless, R. W., Floyd, O. S., Raynes, J. G., Yuen, C. T., and Feizi, T. (1992) classical pentraxins. It is interesting to observe that the long EMBO J. 11, 813– 819 pentraxins do not have the restricted liver inducibility typical 17. Hind, C. R., Collins, P. M., Renn, D., Cook, R. B., Caspi, D., Baltz, M. L., and of CRP and SAP (upon interleukin-6 stimulation) and show a Pepys, M. B. (1984) J. Exp. Med. 159, 1058 –1069 18. Volanakis, J. E. (1982) Ann. N. Y. Acad. Sci. 389, 235–250 more promiscuous pattern of expression in vitro and in vivo. 19. Ying, S. C., Gewurz, A. T., Jiang, H., and Gewurz, H. (1993) J. Immunol. 150, PTX3 can be expressed by endothelial cells, hepatocytes, fibro- 169 –176 blasts, and monocytes in response to lipopolysaccharide and 20. Jiang, H., Siegel, J. N., and Gewurz, H. (1991) J. Immunol. 146, 2324 –2330 21. Jiang, H., Robey, F. A., and Gewurz, H. (1992) J. Exp. Med. 175, 1373–1379 inflammatory cytokine (27, 29) and is induced by lipopolysac- 22. Bristow, C. L., and Boackle, R. J. (1986) Mol. Immunol. 23, 1045–1052 charide in vivo in heart and lung but not in liver (30, 31). 23. Pepys, M. B., Dyck, R. F., de Beer, F. C., Skinner, M., and Cohen, A. S. (1979) Clin. Exp. Immunol. 38, 284 –293 The results reported here show that the long pentraxin PTX3 24. de Beer, F. C., Baltz, M. L., Holford, S., Feinstein, A., and Pepys, M. B. (1981) exhibits structural and functional similarities as well as differ- J. Exp. Med. 154, 1134 –1139 ences when compared with the classical pentraxins CRP and 25. Garcia de Frutos, P., Hardig, Y., and Dahlback, B. (1995) J. Biol. Chem. 270, 26950 –26955 SAP. PTX3 forms multimers as CRP and SAP do, but these 26. Hamazaki, H. (1987) J. Biol. Chem. 262, 1456 –1460 differ in size and structural features (requirement for Cys 27. Breviario, F., d’Aniello, E. M., Golay, J., Peri, G., Bottazzi, B., Bairoch, A., bonds). PTX3 does not recognize the pentraxin ligands (Ca , Saccone, S., Marzella, R., Predazzi, V., Rocchi, M., Della Valle, G., Dejana, E., Mantovani, A., and Introna, M. (1992) J. Biol. Chem. 267, 22190 –22197 PE, PC, HPA) with the exception of C1q. This finding is con- 28. Lee, G. W., Lee, T. H., and Vilcek, J. (1993) J. Immunol. 150, 1804 –1812 sistent with the view that this pentraxin, secreted by macro- 29. Vidal Alles, V., Bottazzi, B., Peri, G., Golay, J., Introna, M., and Mantovani, A. (1994) Blood 84, 3483–3493 phages and endothelial cells following stimulation with inter- 30. Lee, G. W., Goodman, A. R., Lee, T. H., and Vilcek, J. (1994) J. Immunol. 153, leukin-1, tumor necrosis factor, and bacterial components, may 3700 –3707 contribute to the amplification of the effector mechanisms of 31. Introna, M., Vidal Alles, V., Castellano, M., Picardi, G., De Gioia, L., Bottazzi, B., Peri, G., Breviario, F., Salmona, M., De Gregorio, L., Dragani, T. A., innate immunity. In this regard, PTX3 seems to fulfill in tis- Srinivasan, N., Blundell, T. L., Hamilton, T. A., and Mantovani, A. (1996) sues the same function that liver-derived CRP and SAP exert Blood 87, 1862–1872 in the circulation. It remains to be elucidated whether and to 32. Reid, M. S., and Blobel, C. P. (1994) J. Biol. Chem. 269, 32615–32620 33. Noland, T. D., Friday, B. B., Maulit, M. T., and Gerton, G. L. (1994) J. Biol. what extent the observations reported herein for PTX3 can be Chem. 269, 32607–32614 extended to other recently identified long pentraxins. 34. Seery, L. T., Schoenberg, D. R., Barbaux, S., Sharp, P. M., and Whitehead, A. S. (1993) Proc. R. Soc. Lond. Ser. B Biol. Sci. 253, 263–270 Acknowledgments—We are grateful to Drs. Paul Proost and Jo Van 35. Schlimgen, A. K., Helms, J. A., Vogel, H., and Perin, M. S. (1995) Neuron 14, Damme for one of the two microsequence analyses of the purified 519 –526 protein and to Professor Mark B. Pepys for help with the binding assay 36. 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