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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 41, pp. 28263–28275, October 9, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition Received for publication, April 16, 2009, and in revised form, July 13, 2009 Published, JBC Papers in Press, July 24, 2009, DOI 10.1074/jbc.M109.009225 ‡ § ‡ ‡ ¶ § Ying Jie Ma , Andrea Doni , Tina Hummelshøj , Christian Honore´ , Antonio Bastone , Alberto Mantovani , ‡1 Nicole M. Thielens , and Peter Garred From the Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen 2100, Denmark, the Istituto Clinico Humanitas, Rozzano 20089, Italy, the Department of Biochemistry and Molecular Pharmacology, Mario Negri Institute for Pharmacological Research, Milan 20157, Italy, and the Laboratoire d’Enzymologie Mole´culaire, Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075, CNRS-CEA-Universite´ Joseph Fourier, Grenoble 5075, France The long pentraxin 3 (PTX3) is a multifunctional soluble pat- C-terminal fibrinogen-like (FBG) domain involved in innate tern recognition molecule that is crucial in innate immune pro- immune defense (1, 2). In humans, three types of ficolins have tection against opportunistic fungal pathogens such as Aspergil- been identified as follows: Ficolin-1 (M-ficolin), Ficolin-2 (L-fi- lus fumigatus. The mechanisms that mediate downstream colin), and Ficolin-3 (H-ficolin/Hakata antigen). They function effects of PTX3 are largely unknown. However, PTX3 interacts as recognition molecules in the lectin complement pathway with C1q from the classical pathway of the complement. The along with mannose-binding lectin but with differentiated ficolins are recognition molecules of the lectin complement complement activating capacity (3). Ficolin-2 and Ficolin-3 cir- pathway sharing structural and functional characteristics with culate in the blood with a median concentration of 5 and 25 C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, g/ml, respectively (4, 5). Ficolin-2 is mainly produced in the and -3) interact with PTX3 and whether the complexes are able liver, whereas Ficolin-3 is synthesized in both the liver and to modulate complement activation on A. fumigatus. Ficolin-2 lungs, with the highest expression in the lungs (3). Ficolin-1 is could be affinity-isolated from human plasma on immobilized primarily expressed by bone marrow-derived cells and lung epi- PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 thelial cells (3, 6–8) and has recently been shown to be present interacted with PTX3 in a calcium-independent manner. Fico- in the blood with a median plasma concentration of 60 ng/ml lin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus (9). The ficolin genes (FCN1, -2, and -3) are polymorphic, and directly, but this binding was enhanced by PTX3 and vice versa. particularly polymorphisms in FCN2 regulate both the level Ficolin-2-dependent complement deposition on the surface of and function of Ficolin-2 (4, 10, 11). In this respect, a base sub- A. fumigatus was enhanced by PTX3. A polymorphism in the stitution in exon 8 at position 6359 (C3T) causing a threonine FCN2 gene causing a T236M amino acid change in the fibrino- to be replaced by a methionine (T236M) in the FBG domain of gen-like binding domain of Ficolin-2, which affects the binding Ficolin-2 has been shown to cause decreased binding activity to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus toward GlcNAc (10). significantly. These results demonstrate that PTX3 and Fico- Ficolin-1 has been reported to bind to GlcNAc, GalNAc, and lin-2 may recruit each other on pathogens. The effect was alle- sialic acid (8, 12). It may opsonize Staphylococcus aureus via viated by a common amino acid change in the fibrinogen-like GlcNAc and interact with a smooth-type strain of Salmonella domain of Ficolin-2. Thus, components of the humoral innate typhimurium through an unknown ligand, the binding of which immune system, which activate different complement path- is not diminished by GlcNAc (8). Ficolin-2 has been shown to ways, cooperate and amplify microbial recognition and effector recognize specific pathogen-associated molecular patterns, functions. which are typically located in pathogen cell membranes, such as lipoteichoic acid and peptidoglycan in Gram-positive bacteria cell walls, lipopolysaccharide in Gram-negative bacteria cell The ficolins are multimeric collagen-like proteins consisting walls, and 1,3--D-glucan in yeast and fungal cell walls (13, 14). of an N-terminal domain, a collagen-like domain (CD), and a The ligand specificity of Ficolin-2 has also been defined as acetyl groups, including those of N-acetylmannosamine, Glc- NAc, GalNAc as well as acetyl groups on cysteine, glycine, and * This work was supported by the grants from The Benzon Foundation, The Lundbeck foundation, The Carlsberg Foundation, Rigshospitalet, The Cap- choline (15). Ficolin-2 recognizes clinically important patho- ital Region of Denmark, The European Commission (Project Tolerage), gens, like S. typhimurium, S. aureus, and Streptococcus pneu- Telethon, and the Italian Ministry of Health. moniae (13, 16, 17). Ficolin-3 shows affinity for GlcNAc, Gal- To whom correspondence should be addressed. Tel.: 45-35457631; Fax: 45-35398766; E-mail: [email protected]. NAc, and D-fucose and may interact with S. typhimurium, The abbreviations used are: CD, collagen-like domain; BSA, bovine serum Salmonella minnesota, and Aerococcus viridans (17, 18). albumin; CHO, Chinese hamster ovary; CRP, C-reactive protein; ELISA, enzyme-linked immunosorbent assay; FBG, fibrinogen-like domain; G418, geneticin; LPS, lipopolysaccharide; MASP, mannose-binding lec- tin-associated serine protease; MBL, mannose-binding lectin; MTX, acid; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane- methotrexate; PBS, phosphate-buffered saline; FCS, fetal calf serum; 1,3-diol; PTX3, pentraxin 3; HRP, horseradish peroxidase; FACS, fluores- FITC, fluorescein isothiocyanate; MOPS, 4-morpholinepropanesulfonic cence-activated cell sorter. This is an Open Access article under the CC BY license. OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28263 Ficolins and PTX3 The long pentraxin 3 (PTX3) is a soluble pattern recognition Pharmingen. Goat anti-mouse IgG-FITC conjugate, swine anti- molecule mediating innate immune recognition (19). PTX3 is a rabbit IgG-FITC conjugate, and a rabbit polyclonal anti-C4 glycoprotein of 45 kDa, which assembles into an octameric antibody were obtained from Dako (Glostrup, Denmark). structure through protomer linkage by disulfide bonds (20). Restriction enzymes, Hybond ECL nitrocellulose membrane, PTX3 shares C-terminal structural similarity with the classic donkey horseradish peroxidase-conjugated anti-rabbit Ig short pentraxins, C-reactive protein (CRP), and serum amyloid (NAP340V), and streptavidin-horseradish peroxidase conju- P component, whereas the N-terminal sequence differs from gate (RPN 1231) were from Amersham Biosciences. Liver and the other proteins (21). Myeloid cells are a major source of peripheral leukocyte cDNA was from Clontech. pSV neo was PTX3, but PTX3 has also been shown in vitro to be produced by from Clontech. KOD DNA polymerase was from Novagen a variety of cells in response to inflammatory signals (21). Dur- (EMD Biosciences, San Diego). BigDye terminator cycle ing inflammation PTX3 is rapidly up-regulated and released sequencing kit, the model 492 gas-phase sequencer, the model into the surrounding tissue and into the bloodstream. PTX3 140C HPLC system, and the model 610A data analysis software interacts with C1q and participates in activation of the classical package (version 2.1) were from Applied Biosystems (Naerum, complement pathway (22, 23). Moreover, it has also been Denmark). Cell culture utensils were from CM-lab (Vording- shown that PTX3 binds the complement regulatory factor H borg, Denmark). Trypsin, geneticin (G418), hypoxanthine/thy- and that this interaction regulates the alternative pathway of midine media supplement (HT-supplement), L-glutamine solu- complement (24). tion, penicillin/streptomycin solution, methotrexate (MTX), PTX3 can interact with a number of different pathogens, dialyzed fetal bovine serum, Iscove’s modified Dulbecco’s bacteria as well as fungi and viruses. A specific binding has been medium, GlcNAc, mannose, D-galacto-D-mannan, Curdlan observed for selected Gram-positive and Gram-negative bacte- from Alcaligenes faecalis (-1,3-glucan hydrate) (C7821), ria, including S. aureus, Pseudomonas aeruginosa, S. typhi- EDTA, EGTA, bovine serum albumin (BSA), and GlcNAc-aga- murium, Klebsiella pneumoniae, S. pneumoniae, and Neisseria rose were all from Sigma. PowerCHO-1 CD was from Lonza meningitidis (21). PTX3 also binds zymosan and conidia from (Basel, Switzerland). EndoFree plasmid maxi kit was from Qia- Aspergillus fumigatus) (25). Furthermore, it has been shown gen (VWR International A/S, Albertlund, Denmark). Quantum that ptx3 knock-out mice are extremely susceptible to invasive Prep plasmid miniprep kit and the molecular weight standard, pulmonary aspergillosis. The phenotypic defect can be com- Precision prestained protein standard, were from Bio-Rad. pletely reversed by treatment with recombinant PTX3 (25, 26). Escherichia coli One Shot TOP10F Competent Cells, Lipo- These data indicate that PTX3 is important in protection fectamine PLUS reagent kit, NuPAGE 3–8% Tris acetate gel, against A. fumigatus, which has become a major cause of mor- 10% Bistris gel, Tris acetate SDS running buffer, MOPS SDS bidity in medical institutions because of the increasing number running buffer, NuPAGE transfer buffer, and NuPAGE LDS of immunosuppressed patients (27). Sample Buffer were all from Invitrogen. SuperSignal West Based on the knowledge of the structural and functional sim- Femto maximum sensitivity substrate was from Pierce, Bie and ilarities between C1q and the ficolins, this study was designed to Berntsen A/S (Roedovre, Denmark). Microtiter plates (Maxi- characterize a possible interaction between the ficolins and Sorp) were from Nalge Nunc (Roskilde, Denmark). OPD tablets PTX3 using A. fumigatus as a model. Based on our data, we were from Dako (Glostrup, Denmark). Phosphate-buffered propose an important role for previously unlinked collabora- saline (PBS) buffer (10 mM Na HPO , 1.5 mM KH PO , 137 mM 2 4 2 4 tion of PTX3 and Ficolin-2, but not Ficolin-1 and Ficolin-3, in NaCl, 2.7 mM KCl, pH 7.4) was obtained from Bie and Berntsen. the recognition of A. fumigatus and amplification of comple- HEPES buffer (25 mM HEPES, 155 mM NaCl, 5 mM CaCl ,pH ment activation. Moreover, our results demonstrate functional 7.4) was obtained from the hospital pharmacy at Rigshospitalet, consequences of the Ficolin-2 T236M substitution in the inter- Copenhagen, Denmark. Recombinant PTX3, MBL, and action between PTX3 and A. fumigatus. MASP-2 were produced in CHO cells and immunoaffinity-pu- rified under endotoxin-free conditions (25, 28). Natural PTX3 EXPERIMENTAL PROCEDURES from human fibroblast supernatants was purified by ion-ex- Antibodies and Reagents—Mouse monoclonal antibodies change chromatography. A. fumigatus conidia were obtained against human Ficolin-1 (clone FCN115), Ficolin-2 (clone from a clinical isolate (26). Dynabeads Pan mouse IgG was FCN216, clone FCN219, and biotinylated FCN219), and Fico- obtained from Invitrogen. lin-3 (clone FCN334) were characterized in our laboratory (4, 5, Construction of FCN Expression Plasmids—Liver cDNA was 9). Mouse monoclonal antibody against MBL (HYB 131-11) used as a template for PCR amplification for FCN2 and FCN3 was purchased from BioPorto (Gentofte, Denmark). Rabbit and peripheral leukocyte cDNA for FCN1. Oligonucleotide polyclonal anti-Ficolin-1 antibody and biotinylated rabbit poly- primer sets were designed (FCN-1, sense primer 5-ctagtcta- clonal anti-Ficolin-2 and -3 antibody were obtained from gagcgagatggagctgagtggagcca-3 and antisense primer 5-accg- Hycult Biotechnology (HP9039, Uden, The Netherlands) and R gaattccctaggcgggccgcaccttcat-3; FCN-2, sense primer 5- & D Systems, Inc. (BAF2367 and BAF2428, Minneapolis, MN), ctaggtcgacgcgagatggagctggacagagc-3 and antisense primer respectively. A rabbit polyclonal anti-C1q antibody and HRP- 5-accgcccgggcctaggcaggtcgcaccttcat-3; and FCN-3, sense conjugated rabbit anti-mouse IgG were obtained from Dako primer 5-ctaggtcgacgcgagatggatctactgtggatcctg-3 and anti- (Glostrup, Denmark). HRP-conjugated donkey anti-rabbit IgG sense primer 5-accgcccgggcctatcgaagcatcatccgaac-3)to and streptavidin-HRP were purchased from Amersham Bio- obtain an XbaI site at the 5-end and an EcoRI site at the 3-end sciences. Streptavidin-phycoerythrin was purchased from for FCN1 and a SalI site at the 5-end and an SmaI site at the 28264 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 41 •OCTOBER 9, 2009 Ficolins and PTX3 3-end for FCN2 and FCN3, respectively. The amplified prod- performed with SuperSignal West Femto maximum sensitivity ucts were ligated into the mammalian expression vector substrate on autoradiographic films. Precision prestained pro- pEDdC. tein standard (Bio-Rad) was utilized as a molecular weight The Ficolin-2 T236M variant was generated using the standard. QuikChange XL site-directed mutagenesis kit (Stratagene, La Affinity Chromatography—Human EDTA-plasma (25 ml) Jolla, CA) according to the manufacturer’s protocol, using the from healthy donors was diluted in PBS and applied to CNBr- FCN2 ligated-pEDdC cDNA described above as a template. Sepharose beads (Amersham Biosciences) derivatized with One set of oligonucleotides (5-ggagattccctgatgttccacaacaac-3 recombinant PTX3. CNBr-Sepharose beads derivatized with and its reverse complement 5-gttgttgtggaacatcagggaatctcc-3) BSA (Sigma) and recombinant MBL were used as control. The was designed for generating a point mutation at the amino acid unbound material was washed from the column with 10-bed residue at position 236. Constructions were confirmed by Big- column volumes of PBS 0.05% Tween 20 (Merck). The bound Dye terminator cycle DNA sequencing. The final constructions proteins were eluted with 0.1 M glycine-HCl, pH 2.8, immedi- were purified by EndoFree plasmid maxi kit preparation for ately adjusted to pH 7.5 with 1.5 M Tris-HCl, pH 8.8, and col- subsequent transfection. lected directly into a 5-kDa cut-off Vivaspin centrifuge filter Transfection and Protein Expression—The pEDdC vector (Sartorius, Epsom, UK). Fractions (5 g of total proteins) were carries a cloning sequence for insertion of the target gene fol- analyzed by SDS-PAGE and Western blot. lowed by the selectable and amplifiable marker gene dihydro- Binding of Ficolins to Solid Phase PTX3—To determine folate reductase. The expression vectors described above were whether ficolins interact with PTX3, ELISA microtiter plates transfected into the CHO DG44 cell line by using Lipo- were coated overnight at 4 °C with various concentrations of fectamine as described previously (29). Briefly, the untrans- PTX3 (0.3, 1, 3, and 10 g/ml) and BSA (10 g/ml) in coating fected cells were cultured with Iscove’s modified Dulbecco’s buffer (15 mM Na CO ,35mM NaHCO , pH 9.6). All reaction 2 3 3 medium supplemented by HT media supplement (1),2mM volumes were 100 l, and plates were washed after each step in L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, TBS-Ca (20 mM Tris, 150 mM NaCl, 5 mM CaCl , pH 7.4) 10% heat-inactivated FBS at 37 °C in 5% CO , 95% humidity. containing 0.05% (v/v) Tween 20. Plates were incubated for 1 h Cell splitting was conducted with 0.05% trypsin in PBS. Trans- at room temperature with TBST-Ca containing 1% (w/v) fection for obtaining stable cell lines was performed by using BSA to block-exposed wells. Ficolin-1, -2, or -3 dilution series in the Lipofectamine PLUS reagent kit. Transfection was per- TBS-Ca (6.65, 2, 0.6, and 0.2 g/ml) were added to the wells formed by seeding 8 10 cells in 6-cm culture wells. and incubated for5hat37 °C. Bound Ficolin-1, -2, or -3 was The first selection of stable cell line was performed using detected using polyclonal anti-Ficolin-1 antibody, biotinylated G418 to obtain FCN1, FCN2, FCN3, and FCN2-T236M monoclonal anti-Ficolin-2 antibody (clone FCN219), and bio- mutant-positive cells, respectively. The second selection and tinylated monoclonal anti-Ficolin-3 antibody (clone FCN334) gene amplification were conducted with MTX at a final con- by overnight incubation at 4 °C, followed by1hof incubation at centration of 50 nM with G418 supplementation (0.5 mg/ml) 37 °C with HRP-conjugated F(ab) from anti-rabbit donkey and by omitting hypoxanthine and thymidine at the beginning. IgG for Ficolin-1 and HRP-conjugated streptavidin for Fico- When the cell viability recovered, 100 and 200 nM of MTX were lin-2 and -3, respectively. Finally, peroxidase reaction was per- sequentially applied for the subsequent selection. Afterward, formed by using ortho-phenylenediamine/hydrogen peroxi- the stable cell line was utilized to produce recombinant Fico- dase solution as substrate. The reactions were terminated using lin-1, -2, and -3 and Ficolin-2 mutant by culturing in serum-free 1 M H SO , and absorbance was read at 490 nm. 2 4 medium (PowerCHO-1 CD). The concentrations of ficolins in In some experiments, plates were coated with PTX3, fol- serum-free medium were calculated by ELISA from a standard lowed by incubation of Ficolin-1, -2, or -3 in the presence or curve, which was fitted using previously characterized recom- absence of GlcNAc at concentrations ranging from 0 to 300 binant Ficolin-1, -2, and -3 with a His tag produced in our lab- mM, and also with 5 mM of the calcium chelator EGTA. Bound oratory (5, 9, 29). ficolins were detected as depicted above. SDS-PAGE and Western Blots—Proteins were separated by To compare natural ficolins and recombinant ficolins, plates NuPAGE 3–8% Tris acetate gels or 10% Bistris gels under non- were coated with recombinant PTX3, followed by addition of reducing or reducing conditions according to the method of natural and recombinant Ficolin-2 or -3, respectively. Bound Laemmli (30) and stained with Coomassie Brilliant Blue. The ficolins were detected as described above. Alternatively, wells separated proteins were transferred to nitrocellulose (Hybond, coated with natural PTX3 or recombinant PTX3 were incu- ECL, RPN78D, GE Healthcare) using the Xcell II mini-cell blot bated with recombinant Ficolin-1, -2, and -3, respectively. apparatus in NuPAGE transfer buffer. For detection of Fico- Binding of Ficolin-2 to PTX3 Using Surface Plasmon Reso- lin-1, polyclonal anti-Ficolin-1 antibody was applied as primary nance Spectroscopy—Analyses were performed using a BIAcore antibody. For detection of Ficolin-2 and Ficolin-2 T236M, bio- 3000 instrument (GE Healthcare). Recombinant PTX3 was tinylated FCN219 was used as primary antibody. Biotinylated diluted to 100 g/ml in 10 mM sodium formate, pH 3.5, and polyclonal anti-Ficolin-3 antibody was utilized to detect Fico- immobilized on the surface of a CM5 sensor chip (GE Health- lin-3. A rabbit polyclonal antibody was used to detect C1q. In care) using the amine coupling chemistry. Binding of recombi- the subsequent procedure, blots were stained with HRP-conju- nant purified Ficolin-2 (29) to immobilized PTX3 (4000 reso- gated donkey anti-rabbit IgG antibody, HRP-conjugated rabbit nance units) was measured at a flow rate of 20 l/min in 150 mM anti-mouse IgG, or HRP-linked streptavidin. Development was NaCl, 2 mM CaCl ,20mM HEPES, pH 7.4, containing 0.005% OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28265 Ficolins and PTX3 surfactant P20 (GE Healthcare). Regeneration of the surface g/ml) at 37 °C for 1 h. Bound PTX3 was detected as indicated was achieved by injection of 10 lof1 M sodium acetate, pH 7.2 above. Alternatively, PTX3 was incubated with A. fumigatus at followed by 10 lof10mM NaOH. Equivalent volumes of each 37 °C for 1 h prior to incubation with Ficolin-2. Binding of Fico- protein sample were injected over an activated-deactivated sur- lin-2 in the presence of PTX3 was also measured using the same face to serve as blank sensorgrams for subtraction of the bulk method. refractive index background. In some experiments, A. fumigatus conidia were incubated Data were analyzed by global fitting to a 1:1 Langmuir bind- with Ficolin-2 T236M instead of wild type Ficolin-2. Ficolin-2 ing model of both the association and dissociation phases for six T236M binding was detected with monoclonal anti-Ficolin-2 concentrations simultaneously, using the BIAevaluation 3.2 antibody FCN219 by incubation at 4 °C for 30 min and meas- software (GE Healthcare). The apparent equilibrium dissocia- ured by FACS analysis. tion constants (K ) were calculated from the ratio of the disso- C4 Deposition on A. fumigatus—C4 deposition on A. fumiga- ciation and association rate constants (k /k ). The molar con- tus was assessed by FACS analysis. As source of complement off on centration of Ficolin-2 was estimated assuming a tetrameric component, C1q-deficient serum was used in subsequent structure of 414 kDa (12 polypeptide chains of 34.5 kDa each) experiments. C1q-deficient serum was obtained from a C1q- (29). deficient individual with normal levels of Ficolin-2 and MBL Binding of Wild Type Ficolin-2 and Its Mutant to GlcNAc (31). Subsequently, MBL was depleted by agitating the C1q- Beads—Three g of wild type Ficolin-2 or Ficolin-2 T236M deficient serum with mannan-agarose beads at 4 °C for 2 h. To were incubated with 50 l of GlcNAc-agarose beads overnight deplete serum Ficolin-2, magnetic pan-mouse IgG Dynabeads with continuous stirring at 4 °C. Total reaction volume was was applied according to the manufacturer’s instructions. In made up to 1.5 ml with TBS-Ca as binding buffer. Following brief, Ficolin-2 was depleted by incubation of the MBL-de- centrifugation at 1000 rpm for 1 min, the beads were washed pleted and C1q-deficient serum with FCN219 (10 g/ml) and three times with binding buffer. Bound proteins were eluted Dynabeads at 4 °C with rotation. 20% C1q-deficient serum with 0.4 M GlcNAc in binding buffer. The concentration of wild depleted of MBL (C1q /MBL serum) and Ficolin-2 depleted type Ficolin-2 and Ficolin-2 T236M in eluates was measured by (C1q /MBL, Ficolin-2 serum) was prepared by dilution depleted ELISA as described above. of the C1q /MBL serum in HEPES buffer. depleted Binding of Ficolin-1, -2, and -3 to A. fumigatus—A. fumigatus For C4 deposition, A. fumigatus conidia were incubated with conidia were washed and resuspended in 100 l of HEPES 20% C1q /MBL serum with supplementary C4 (10 depleted buffer containing 1% heat-inactivated FCS at 2.4 10 cells/ml g/ml) at 37 °C for 1 h. Alternatively, 20% C1q /MBL, Ficolin- and incubated with Ficolin-1, -2, or -3 at 37 °C for 1 h. All 2 serum reconstituted by Ficolin-2 (7 g/ml) was incu- depleted reaction volumes were 100 l, and A. fumigatus conidia were bated with A. fumigatus conidia with supplementary C4 (10 washed after each step in HEPES buffer containing 1% heat- g/ml) at 37 °C for 1 h. All reaction volumes were 100 l, and A. inactivated FCS. Bound proteins were detected with mono- fumigatus was washed after each step in HEPES buffer contain- clonal anti-Ficolin-1 antibody (clone FCN115), anti-Ficolin-2 ing 1% heat-inactivated FCS. C4 deposition was detected with a antibody (clone FCN219), or anti-Ficolin-3 antibody (clone rabbit anti-human C4 antibody by incubation at 4 °C for 30 FCN334) by incubation at 4 °C for 30 min, respectively, fol- min, followed by incubation with swine anti-rabbit IgG FITC lowed by 15 min of incubation at 4 °C with FITC-conjugated conjugate at 4 °C for 15 min. Finally, C4 deposition was assessed anti-mouse IgG from goat. Finally, the A. fumigatus conidia by FACS analysis, and data were analyzed by CellQuest Pro were washed and resuspended in 200 l of HEPES buffer con- software (BD Biosciences). taining 1% heat-inactivated FCS. Flow cytometry was carried In some experiments, A. fumigatus conidia were preincu- out on a FACSCalibur (BD Biosciences), and data were ana- bated with 10 g/ml PTX3 at 37 °C for 1 h prior to assessment lyzed by CellQuest Pro software (BD Biosciences). of C4 deposition. As a control, MBL levels in C1q-deficient In some experiments, A. fumigatus conidia were incubated serum and C1q /MBL serum were determined by depleted with Ficolin-2 in the presence of GlcNAc (0.3 M), mannose (0.3 assessing MBL binding to A. fumigatus. A. fumigatus conidia M), EDTA (20 mM), Curdlan (-1,3-glucan hydrate) (250 were incubated with 20% C1q /MBL serum at 37 °C for depleted g/ml), or a combination of GlcNAc (0.3 M) and Curdlan (250 1 h. Bound MBL was detected with anti-MBL monoclonal anti- g/ml). Ficolin-2 binding was detected as described above. body (HYB 131-11) and goat anti-mouse IgG FITC conjugate PTX3 Binding to A. fumigatus—To verify PTX3 binding to A. by incubation at 4 °C for 30 and 15 min, respectively. Further- fumigatus, PTX3 was incubated with A. fumigatus at a final more, Ficolin-2 levels in C1q /MBL, Ficolin-2 serum depleted concentration of 10 g/ml in the absence or presence of 20 were determined by Western blot. C1q /MBL serum depleted g/ml galactomannan at 37 °C for 1 h, followed by incubation before and after depletion (5 g of total proteins) was analyzed of biotinylated anti-PTX3 monoclonal antibody at 4 °C for by SDS-PAGE under reducing conditions, followed by detec- 30 min. PTX3 binding was detected by incubation of strepta- tion of biotinylated rabbit polyclonal anti-Ficolin-2 antibody vidin/phycoerythrin at 4 °C for 15 min and measured by and streptavidin-HRP, respectively. As a supplementary exper- FACS analysis. iment, recombinant Ficolin-2 (7.0 g/ml) and recombinant Interaction between Ficolin-2 and PTX3 on A. fumigatus— MASP-2 (1.0 g/ml) were applied as substitute complement The procedure of Ficolin-2 binding to A. fumigatus was the initiators instead of serum. same as mentioned above. A. fumigatus was incubated with Statistical Analysis—Data represent the means S.D. or Ficolin-2 (7 g/ml), followed by incubation with PTX3 (10 S.E. of at least three independent experiments. The means 28266 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 41 •OCTOBER 9, 2009 Ficolins and PTX3 FIGURE 1. Ficolin-2 isolation from human plasma by immobilized PTX3. A CNBr-Sepharose column coupled by BSA (1) or recombinant PTX3 (2) was used. Western blot analysis of reduced Ficolin-2 (A) and C1q (B) were per- formed on eluted fractions (5 g of total proteins/lane). Recombinant MBL- attached Sepharose columns were also passed with human plasma. Ficolin-2 (C) and C1q (D) in loading material (lane 3), pass-through (lane 2), and eluate (lane 1) were detected by SDS-PAGE followed by Western blot. S.D. were calculated by Excel software (Microsoft). Student’s t test was used to calculate significance, and p 0.05 was con- sidered to represent a statistical significant difference between two sample means. RESULTS Ficolin-2 Isolation from Human Plasma by Sepharose-PTX3 Beads—In ligand fishing experiments of human plasma involv- ing PTX3 affinity chromatography coupled to mass spectrom- etry, several serum proteins such as C1q, factor H, properdin, MASPs, and Ficolin-2 were found to potentially interact to immobilized PTX3. Analysis with SDS-PAGE and subsequent Western blot was conducted on the fraction eluted from Sepha- rose-PTX3 beads incubated with human plasma. In Fig. 1, rep- resentative experiments with Ficolin-2 and C1q are shown. Sepharose-BSA and MBL beads were used as controls. Recombinant Ficolin-1, -2, and -3 Characterization—Re- combinant ficolins without His tag were expressed by using a CHO cell expressing system. The cDNA of FCN1, -2, and -3 was cloned and inserted into the pEDdC expression vector contain- FIGURE 2. Oligomerization of Ficolin-1, -2, and -3 expressed in CHO cells. ing a dihydrofolate reductase expression site for MTX-induced Ficolin-1 (A), Ficolin-2 (B), and Ficolin 3 (C) produced in CHO cells were sub- amplification. CHO DG44 cell lines were transfected with the jected to 3– 8% Tris acetate SDS-PAGE under nonreducing (lane 1) or reducing (lane 2) conditions and analyzed by Western blot. The multimeric pattern of expression vectors containing the FCN1, -2, and -3 insert along recombinant Ficolin-1 (lane 1, D), Ficolin-2 (lane 1, E), and Ficolin-3 (lane 1, F) with pSV containing a neomycin resistance site and 2neo was compared with monocyte-derived Ficolin-1 (lane 2, D), serum Ficolin-2 (lane 2, E), and serum Ficolin-3 (lane 2, F). selected with G418. Clones expressing ficolins from G418 selection were generated through multicycle of amplification with elevated concentration of MTX. Using ELISA, the concen- -2, and -3 presented multimeric assembly laddered with 10 trations of Ficolin-1, -2, and -3 in the culture supernatants were bands (lane 1 in Fig. 2, A–C) but ran as a single band of34, 35, quantified as 12, 7, and 10 g/ml, respectively. To determine and 34 kDa under reducing conditions on Western blot, respec- the oligomerization state of ficolins, recombinant and natural tively (lane 2 in Fig. 2, A–C). The multimeric pattern is in cor- Ficolin-1, -2, and -3 were analyzed by 3–8% Tris acetate SDS- respondence with the pattern seen in a serum pool (Fig. 2, D–F). PAGE under nonreducing or reducing conditions, followed by This result showed that CHO cells secreted recombinant Fico- Western blot (Fig. 2). Under nonreducing conditions, Ficolin-1, lin-1, -2, and -3 were oligomerized in subunits stabilized via OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28267 Ficolins and PTX3 FIGURE 3. Binding of Ficolin-1, -2, and -3 to PTX3. Microtiter wells were FIGURE 4. Effect of GlcNAc on the binding of Ficolin-1, -2, or -3 to PTX3. coated with various concentrations of PTX3 or BSA (10 g/ml) and incubated Microtiter wells were coated with increased concentrations of PTX3 and incu- with the indicated concentrations of Ficolin-1 (A), Ficolin-2 (B) or Ficolin-3 (C) bated with Ficolin-1 (6 g/ml) (A), Ficolin-2 (5.4 g/ml) (B), or Ficolin-3 (6 by two-dimensional serial dilutions. Results are represented as mean S.E. g/ml) (C) in the presence of the indicated concentrations of GlcNAc. Results from at least six independent experiments. are representative of two independent experiments. disulfide bonds, ranging from two to more than four subunits in Ficolin-1 exhibited better binding to PTX3 than Ficolin-3. To a similar fashion to natural proteins. further determine the binding specificity of Ficolin-1, -2, and Binding of Ficolin-1, -2, and -3 to PTX3—To determine -3, we next examined their binding to immobilized PTX3 in the whether Ficolin-1, -2, or -3 binds to PTX3, ELISA wells coated presence of GlcNAc (Fig. 4, A–C). Inclusion of GlcNAc in the with various concentrations of PTX3 were incubated with var- binding buffer attenuated the binding of Ficolin-1 and -2 to ious concentrations of each ficolin. Ficolin-1, -2, and -3 bound immobilized PTX3, although no difference in Ficolin-3 binding to immobilized PTX3 in a dose-dependent manner (Fig. 3, was detected in the presence or absence of GlcNAc. These A–C). A substantial binding of Ficolin-2 to PTX3 was observed, results implied that Ficolin-2, in particular, but also Ficolin-1 whereas Ficolin-1 and -3 bound relatively weaker to PTX3. bind to immobilized PTX3 via the FBG domain but not Fico- 28268 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 41 •OCTOBER 9, 2009 Ficolins and PTX3 FIGURE 6. Ficolin-2 interaction with PTX3 using plasmon resonance spec- troscopy. Analysis by surface plasmon resonance spectroscopy of the inter- action of recombinant Ficolin-2 with immobilized PTX3. A, 60l of Ficolin-2 at varying concentrations (bottom to top curves as follows: 1.5, 3, 6, 12, 18, and 24 nM) were injected over 4000 resonance units (RU) of immobilized PTX3 in 150 mM NaCl, 2 mM CaCl , and 20 mM HEPES, pH 7.4, containing 0.005% (v/v) surfactant P20 at a flow rate of 20 l/min. B, 60 l of ficolin-2 at a concentra- tion of 24 nM were injected over immobilized PTX in the running buffer con- taining either 2 mM CaCl or2mM EDTA. The specific signal shown was FIGURE 5. Effect of calcium chelation on the binding of Ficolin-1 and Fico- obtained by subtracting the background signal obtained by injection of the lin-2 to PTX3. Microtiter wells were coated with increasing concentrations of protein sample over the reference surface with no protein immobilized. PTX3 and incubated with Ficolin-1 (6g/ml) (A) and Ficolin-2 (5.4g/ml) (B)in Results are representative from two independent experiments, using differ- the presence of 5 mM EGTA and the indicated concentrations of GlcNAc. ent sensor chips. Results are representative of two independent experiments. Ficolin-2 showed vigorous binding to recombinant PTX3 in a lin-3. We then removed calcium by adding a buffer containing 5 similar manner (Fig. 7A). However, natural Ficolin-3 presented mM EGTA in experiments, including Ficolin-1 and Ficolin-2 no binding compared with slight binding of recombinant Fico- (Fig. 5, A and B). These experiments resulted in a substantial lin-3 (Fig. 7A). Subsequently, we also evaluated binding of increase in binding of both Ficolin-1 and Ficolin-2 to PTX3, but recombinant ficolins to natural and recombinant PTX3. ELISA GlcNAc could still inhibit the interactions. wells coated with natural or recombinant PTX3 were incubated The ability of recombinant Ficolin-2 to interact with PTX3 with each recombinant ficolin. Recombinant Ficolin-2 bound was also investigated by surface plasmon resonance spectros- both natural and recombinant PTX3; however, the binding to copy. As shown in Fig. 6, Ficolin-2 bound to immobilized PTX3 natural PTX3 is dominant, approximately three times higher in the presence of 2 mM CaCl . The kinetic parameters of the than to recombinant PTX3 (Fig. 7B). Furthermore, recombi- interaction were determined by recording sensorgrams at vary- nant Ficolin-1 and -3 presented very low binding to both natu- ing ficolin concentrations (Fig. 6A) and evaluating the data by ral and recombinant PTX3 (Fig. 7B). No signal was detected in global fitting as described under “Experimental Procedures.” A BSA-coated wells (Fig. 7). 5 1 1 3 1 k value of 6.0 10 M s and a k value of 3.9 10 s Binding of Ficolin-2 to A. fumigatus—To further provide evi- on off were determined, yielding a resulting apparent K value of 6.5 dence that the observed interaction between ficolins and PTX3 nM, indicative of a high affinity interaction. Binding was main- may be physiologically relevant, we focused our attention on A. tained when EDTA was substituted for Ca in the running fumigatus as an anchored matrix. To assess interaction of buffer, and the complex appeared even more stable, as reflected the ficolins with PTX3 on A. fumigatus, we first examined by the slower dissociation phase (Fig. 6B). whether ficolins by themselves were able to bind to the surface Relative Binding of Natural and Recombinant Ficolins to of A. fumigatus by FACS analysis. PTX3—To determine binding capacity of recombinant ficolins As shown in Fig. 8, A and B, we found that Ficolin-2 did bind to PTX3 compared with ficolins from natural material, ELISA to A. fumigatus directly in a dose-dependent manner, but nei- wells coated with recombinant PTX3 were incubated with each ther Ficolin-1 nor -3 did. As a control, binding of recombinant natural or recombinant ficolins. Both natural and recombinant MBL was also assessed concurrently. The binding of Ficolin-2 OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28269 Ficolins and PTX3 PTX3 increased Ficolin-2 binding to A. fumigatus significantly (p 0.05) (Fig. 9B) and vice versa (p 0.01) (Fig. 9C). Enhancement of Ficolin-2-induced C4 Deposition on A. fumigatus by PTX3—To further characterize the physiological relevance of the Ficolin-2-PTX3 interaction on A. fumigatus, we investigated its effect on Ficolin-2-dependent complement pathway activation as assessed by C4 deposition using C1q / MBL serum. MBL depletion was assessed by analysis of depleted MBL binding to A. fumigatus, compared with control reaction without MBL (Fig. 10A). Furthermore, binding of serum Fico- lin-2 to A. fumigatus was also confirmed using C1q / MBL serum (Fig. 10B) before testing C4 deposition, depleted showing that serum-derived and recombinant Ficolin-2 have the same binding pattern to A. fumigatus. Next, we focused our attention on C4 deposition using the C1q /MBL serum depleted verified above. In C1q /MBL serum we could observe depleted deposition of C4 in the absence of PTX3 (Fig. 10C). However, this deposition was significantly increased in the presence of PTX3 (p 0.05), compared with control experiment without PTX3 (Fig. 10C). To verify the above implication regarding the effect of PTX3/ Ficolin-2 interaction on C4 deposition, Ficolin-2 was depleted from C1q /MBL serum and assessed by Western blot as depleted described under “Experimental Procedures” (Fig. 10D, inset), followed by assessment of C4 deposition on A. fumigatus.As shown in Fig. 10D, C4 deposition was reinforced when C1q / FIGURE 7. Binding of ficolins to PTX3 from natural source. A, microtiter wells coated with recombinant PTX3 or BSA (5 g/ml) and incubated with MBL, Ficolin-2 serum was reconstituted by addition of depleted recombinant Ficolin-2 and -3 or serum Ficolin-2 and -3 (5 g/ml). Bound Fico- Ficolin-2. Consistently, this deposition was significantly lin-2 and -3 were detected. B, microtiter wells coated with recombinant PTX3, increased in the presence of PTX3 (p 0.05), compared with natural PTX3, or BSA (5 g/ml) and incubated with recombinant Ficolin-1, -2, and -3 (5 g/ml). Bound Ficolin-1, -2, and -3 were detected. Data represent control experiment without PTX3 (Fig. 10D). means S.D. from triplicate experiments. Results are representative from To further provide evidence for the preceding results, C1q / two independent experiments that yield similar results. MBL serum was replaced with a more simple system, depleted to A. fumigatus was significantly inhibited in the presence of 0.3 recombinant Ficolin-2 and MASP-2 as substitute complement M GlcNAc (Fig. 8C). Excess mannose or EDTA had no signifi- initiators in the presence of exogenous C4. As shown in Fig. cant effect on Ficolin-2 binding to A. fumigatus (Fig. 8C). In 10E, additional Ficolin-2-MASP-2 complex leads to C4 deposi- contrast, binding of MBL to A. fumigatus was drastically tion on A. fumigatus. Consistent with the above results, PTX3 decreased in the presence of GlcNAc or mannose or EDTA significantly enhanced Ficolin-2-MASP-2 complex-induced C4 (data not shown). deposition on A. fumigatus (p 0.05), compared with control Recognition of -1,3-Glucan on A. fumigatus—Ficolin-2 can experiment without PTX3 (Fig. 10E). recognize -1,3-glucan, a major cell wall component of yeast Reduced Ficolin-2 T236M Binding to GlcNAc, PTX3, and A. and fungi (14), and cell wall analysis of A. fumigatus has shown fumigatus—Previously we found an allelic variant in the FCN2 that -1,3-glucan is extensively distributed on its surface (32). gene that replaced a threonine with a methionine at amino acid To further clarify whether the binding of Ficolin-2 is brought position 236 (Ficolin-2 T236M), which when purified from about by recognition of -1,3-glucan on A. fumigatus,we serum exhibited reduced binding capacity to GlcNAc com- attempted to inhibit the binding of Ficolin-2 using Curdlan pared with wild type Ficolin-2 (10). To clarify whether recom- (-1,3-glucan hydrate). As shown in Fig. 8C, the binding of binant Ficolin-2 T236M also exhibited altered or decreased Ficolin-2 was impaired in the presence of Curdlan (250 g/ml). ligand-binding capacity, an expression vector coding for the Moreover, the combination of GlcNAc and Curdlan drastically Ficolin-2 T236M was generated by site-directed mutagenesis, inhibited the binding of Ficolin-2 (Fig. 8C), suggesting that and the mutated protein was expressed in CHO cells. SDS- Ficolin-2 bound to A. fumigatus in a GlcNAc- and Curdlan-de- PAGE followed by Western blot analysis showed that the pendent manner. protein had a multimeric pattern comparable with that of the Collaboration of Ficolin-2 and PTX3 on A. fumigatus—To wild type protein (Fig. 11A). Moreover, binding of Ficolin-2 address whether an interaction between Ficolin-2 and PTX3 T236M to previously substantiated ligands GlcNAc, PTX3, may occur on the surface of A. fumigatus, we assessed binding and A. fumigatus was assessed by ELISA and FACS analysis, by FACS analysis and compared the reactions in the presence of respectively, as described under “Experimental Procedures.” either PTX3 or Ficolin-2 alone or together (Fig. 9, A–C). As Differential binding capacity was detected between wild type shown in Fig. 9, B and C, Ficolin-2 bound to A. fumigatus much Ficolin-2 and its T236M mutant. In accordance with our better than PTX3, but most interestingly preincubation of previous data, recombinant Ficolin-2 T236M showed signif- 28270 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 41 •OCTOBER 9, 2009 Ficolins and PTX3 FIGURE 8. Binding of ficolins to A. fumigatus. A, binding of ficolins to A. fumigatus. Ficolin-1 (12 g/ml), -2 (6 g/ml), or -3 (10 g/ml) was incubated with A. fumigatus (2.4 10 cells) at 37 °C and detected with anti-ficolin antibodies in flow cytometry. Fluorescence intensity (FL1-H) was plotted against cell number. Results are representative of at least six individual experiments. B, dose-dependent binding of Ficolin-2 to A. fumigatus. Experimental conditions are as indicated in A. C, binding specificity of Ficolin-2 to A. fumigatus. A. fumigatus was incubated with Ficolin-2 (5.5 g/ml) alone or in the presence of EDTA (10 mM), mannose (0.3 M), GlcNAc (0.3 M), Curdlan (250 g/ml), or a combination of GlcNAc and Curdlan, respectively. The mean fluorescence intensity (MFI) was used as assessment of Ficolin-2 binding in B and C. Results are represented as mean S.D. from triplicate experiments. Asterisk refers to the statistical significance versus controls, *, p 0.01. icantly lower binding capacity toward GlcNAc-agarose that both Ficolin-1 and Ficolin-2 interact with CRP and that beads (p 0.05) (Fig. 11B) compared with wild type, as was this complex may mediate complement killing of P. aeruginosa the binding toward immobilized PTX3 and A. fumigatus (35, 36). We have demonstrated that circulating plasma-de- (p 0.05) (Fig. 11, C and D). rived Ficolin-2 interacted with immobilized PTX3 in affinity chromatography experiments. This is similar to what was pre- DISCUSSION viously shown in the case of C1q in binding experiments (22). Moreover, Ficolin-2 and Ficolin-1 were able to bind to PTX3 It is well described that C1q interacts with the short pen- traxin family members CRP and serum amyloid P component adsorbed to microtiter wells in a dose-dependent fashion. and that this complex formation may mediate classical pathway These interactions were partially inhibited by GlcNAc. We also observed some binding with Ficolin-3, but we were not able to complement activation (33, 34). The same has been shown for inhibit the binding with GlcNAc and interpret this result as a solid phase bound PTX3 (22, 23). This complex triggers activa- tion of the classical pathway of complement via the globular nonspecific interaction. Zhang et al. (36) showed that at pH 7.4 heads of C1q. an increased calcium concentration dramatically inhibited the Whether the lectin pathway initiators may have similar func- CRP/Ficolin-2 interaction, indicating that calcium under nor- tions is largely unknown. However, it has recently been shown mal conditions prevents the CRP/Ficolin-2 interaction. Our OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28271 Ficolins and PTX3 ating in the presence of calcium. However, calcium chelation with 5 mM EGTA led to a substantial increase in the binding of Ficolin-2, and especially of Ficolin-1. Accordingly, surface plas- mon resonance spectroscopy experiments showed an increased stability of the PTX3-Ficolin-2 interaction in the presence of EDTA. The increased binding observed by ELISA could still be inhibited with GlcNAc indicating that it was not because of nonspecific trapping in the absence of calcium. It is difficult to relate these findings directly to a physiological situation, but it indicates that the interaction between Ficolin-1, Ficolin-2, and PTX3 is calcium-independent. However, use of a dilution buffer without calcium added yielded the same interaction pat- tern as in the presence of 5 mM calcium (data not shown). Thus, we choose to continue the experiments using calcium-contain- ing buffers, which are closer to physiological conditions. How- ever, according to our results using natural and recombinant proteins, it should be noted that natural Ficolin-2 and recom- binant Ficolin-2 behave in a similar fashion and that natural PTX3 had a higher binding capacity to recombinant Ficolin-2, compared with recombinant PTX3. On the contrary, natural Ficolin-3 showed negligible binding to both natural and recom- binant PTX3 compared with slight binding of recombinant Ficolin-3. These results indicated that the interaction between native PTX3 and native Ficolin-2 in vivo is highly significant and suggest that natural PTX3 and Ficolin-2 might cause a vig- orous interaction in vivo. Nevertheless, because of very low lev- els of Ficolin-1 in the serum (9), we could not perform the corresponding experiments concerning serum versus recombinant Ficolin-1. Furthermore, in our previous reports (20, 37), we have described the folding and sugar composi- tion of recombinant and natural PTX3, indicating no signif- icant differences between PTX3 from natural material or CHO expression system. Infection with A. fumigatus has become an increasing health problem in immunocompromised patients (27). PTX3 has been shown to be critical in the protection against A. fumigatus (25, 26), and Ficolin-2 has been shown to bind to -1,3-glucan (14), which is a major constituent of many fungi. This prompted us to investigate whether the ficolins and PTX3 could cooperate at the surface of A. fumigatus. First, we established by flow cytom- etry that conidia of A. fumigatus served as a very good ligand for Ficolin-2. However, no binding could be observed for either Ficolin-1 or Ficolin-3. Consistent with previous reports, we confirmed specific binding of MBL to A. fumigatus (38) that could be inhibited by GlcNAc, mannose, or EDTA (data not shown). Unlike MBL, the binding of Ficolin-2 to A. fumigatus was not decreased in the presence of mannose or EDTA, but it FIGURE 9. Ficolin-2/PTX3 cross-talk on A. fumigatus. A, binding of PTX3 to was reduced to 50% in the presence of GlcNAc. The latter A. fumigatus assessed by flow cytometry. Fluorescence intensity (FL2-H) was plotted against cell number. B, interaction of Ficolin-2 with PTX3. A. fumigatus finding suggests that more than one binding site in the FBG was incubated with Ficolin-2 alone or preincubated with PTX3 before addi- domain of Ficolin-2 is involved in the interaction with A. tion of Ficolin-2. C, interaction of PTX3 with Ficolin-2. A. fumigatus was incu- bated with PTX3 alone or preincubated with Ficolin-2 before addition of fumigatus. Recently, the crystal structure of the FBG domain of PTX3. The mean fluorescence intensity (MFI) was used as assessment of PTX3 ficolins has been solved and has revealed the location of the or Ficolin-2 binding. Asterisks refer to the statistical significance versus con- major ligand-binding sites of Ficolin-2 (39). MBL has only one trols: *, p 0.05; **, p 0.01. Results shown are mean S.D. and are repre- sentative of at least three independent experiments. specific binding site in the CRD domain to recognize C3-OH and C4-OH of terminal carbohydrate residues by a typical experiments were usually performed at pH 7.4 at a calcium hydrogen bonding interaction (40). In addition to a common concentration of 5 mM showing that the interaction between external S1-binding site that it shares with Ficolin-1 and Fico- PTX3 and Ficolin-1 and in particular Ficolin-2 could be oper- lin-3, Ficolin-2 has three additional binding sites (S2–S4) in the 28272 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 41 •OCTOBER 9, 2009 Ficolins and PTX3 FIGURE 10. C4 deposition depends on Ficolin-2 and Ficolin-2/PTX3 collaboration. A, MBL binding to A. fumigatus in C1q-deficient serum and in MBL- depleted C1q-deficient serum. B, binding of Ficolin-2 in MBL-depleted C1q-deficient serum and of recombinant Ficolin-2 (5.5 g/ml) to A. fumigatus. Fluores- cence intensity (FL1-H) was plotted against cell number in A and B and is representative of at least three independent experiments. C, C4 deposition induced by interaction of Ficolin-2 and PTX3 in MBL-depleted and C1q-deficient serum on A. fumigatus. D, Ficolin-2, MBL-depleted and C1q-deficient serum was reconstituted by addition of recombinant Ficolin-2 and induced C4 deposition in the presence of PTX3 on A. fumigatus. Depletion of Ficolin-2 from MBL- depleted and C1q-deficient serum analyzed by Western blot is shown in the inset. Lane 1, before depletion; lane 2, after depletion. E, reconstruction of PTX3-Ficolin-2-MASP-2 complex-induced C4 activation on A. fumigatus using recombinant MASP-2. The mean fluorescence intensity (MFI) was used to assess C4 deposition. Asterisk indicates the statistical significance versus controls: *, p 0.05. Results represent mean S.D. of four independent experiments. FBG domain (39). These findings explain why Ficolin-2 harbors sites are distant from the Ca -binding site in the Ficolin-2 the capacity of versatile recognition of a variety of acetylated structure, A. fumigatus recognition is not sensitive to EDTA. and carbohydrate-containing compounds. Most of the acety- PTX3 clearly bound A. fumigatus as has been shown before lated ligands bind to sites S2 and S3 of the FBG domain of (25, 26), but to a lesser degree than Ficolin-2. Of particular Ficolin-2, whereas the binding of -1,3-glucan involves the interest was the observation that PTX3 could significantly aug- edge of site S3 and S4 via water-mediated hydrogen bonding ment the deposition of Ficolin-2 on the surface of A. fumigatus. interactions. However, the S3-binding site is shared by both the However, a possibly more intriguing finding was the observa- acetyl group of GlcNAc and the first glucose residue of an elon- tion that Ficolin-2 by itself could enhance the binding of PTX3 gated -1,3-glucan molecule (39). Because the cell membrane to A. fumigatus. This indicates that PTX3 also interacts with of A. fumigatus is composed mainly of chitin (a polymer of -1, Ficolin-2 at sites that are not involved in Ficolin-2 interaction 4-N-acetyl-glucosamine), -1,3-glucan, and galactomannan with A. fumigatus per se. This may also explain the partial inhi- (41), it is reasonable to hypothesize that two kinds of recogni- bition of the interaction between Ficolin-2 and PTX3 that we tion sites are involved in the binding of Ficolin-2 to A. fumiga- observed with GlcNAc on the solid phase polystyrene matrix. tus, which may both include a GlcNAc and a -1,3-glucan To further elucidate whether the Ficolin-2/PTX3 interactions moiety. Our competition experiments using Curdlan (a -1,3- with A. fumigatus may be of physiological relevance, we inves- Glucan hydrate from A. faecalis) suggest that the FBG domain tigated their influence on complement activation induced by of Ficolin-2 preferentially recognizes the -1,3-glucan moiety Ficolin-2. We could show that Ficolin-2 and PTX3 collaborate in addition to the GlcNAc moiety on A. fumigatus, which to boost Ficolin-2-mediated complement deposition. Thus, it is accounts for the strong combined inhibiting effect of the two tempting to speculate that this interaction may play a signifi- compounds. In agreement with the fact that the ligand-binding cant physiological role. OCTOBER 9, 2009• VOLUME 284 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28273 Ficolins and PTX3 microorganisms but also between Ficolin-2 and PTX3. Based on the crystal structure of Ficolin-2 (39), the T236M polymorphism does not directly influence the four putative bindings sites in the FBG domain. Thus, it is more likely that the resulting T236M change in the amino acid composition may affect the tertiary structure of the FBG domain of Ficolin-2, which may have an indirect effect of the binding and interaction properties of the protein. Alternatively, additional binding pockets may exist in the FBG domain of Ficolin-2 not resolved by the present crystallo- graphic observations. In summary, plasma Ficolin-2 could be purified with immobilized PTX3. Ficolin-1 and in particular Ficolin-2 but not Ficolin-3 inter- acted with PTX3 in a calcium-inde- pendent manner. Only Ficolin-2 bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-mediated FIGURE 11. Ligand binding disability of the Ficolin-2 T236M variant. A, multimerization of Ficolin-2 T236M. Wild type (WT) Ficolin-2 (lane 1) and Ficolin-2 T236M variant (lane 2) were analyzed by 10% SDS-PAGE followed complement deposition on the sur- by Western blot under nonreducing (left) or reducing (right) conditions. B, binding of wild type and Ficolin-2 face of A. fumigatus was enhanced T236M to GlcNAc-agarose beads. Ficolin-2 was incubated with GlcNAc-agarose beads and recovered by elu- by PTX3. These results demon- tion with GlcNAc, respectively. The concentration of Ficolin-2 in the eluates was assessed by ELISA. The amount of bound protein (left y axis) and the recovery yield (right y axis) were calculated from ELISA measurements. strate that PTX3 and Ficolin-2 may Results represent mean S.D. of three independent experiments. C, binding of wild type and Ficolin-2 T236M recruit each other on pathogens and to PTX3 in ELISA. Microtiter wells coated with PTX3 or BSA as negative control were incubated with wild type (2.0 g/ml) or Ficolin-2 T236M (2.0 g/ml), and binding was detected with an anti-Ficolin-2 antibody. Results enhance complement activation. are representative of three independent experiments that yielded similar results. D, binding of wild type and This effect was dramatically reduced Ficolin-2 T236M to A. fumigatus in flow cytometry. A. fumigatus was incubated with Ficolin-2. and binding was by a common amino acid change in detected with an anti-Ficolin-2 antibody. The mean fluorescence intensity (MFI) was used to assess Ficolin-2 binding. Asterisks refer to the statistical significance versus controls: *, p 0.05. Results represent mean S.D. the FBG domain of Ficolin-2. Thus, of three independent experiments. components of the humoral innate immune system, which activate dif- No deficiency state of Ficolin-2 in humans has been ferent complement pathways, cooperate and amplify microbial described so far, but promoter variants in the Ficolin-2 gene recognition and effector functions. (FCN2) are associated with differences in the serum concentra- tions (4, 10). However, the GlcNAc binding ability of Ficolin-2 Acknowledgment—We thank Vibeke Weirup for excellent technical is hampered by a polymorphism situated in exon 8 in the FCN2 assistance. gene causing a threonine to be replaced with methionine (T236M) (10). This observation was based on binding studies REFERENCES performed with Ficolin-2 in whole serum. To bring the under- standing of this observation a step further, we produced the 1. Endo, Y., Matsushita, M., and Fujita, T. (2007) Immunobiology 212, 371–379 variant in a recombinant form as we have done with wild type 2. Runza, V. 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Published: Oct 1, 2009