Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition*

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.

The ficolins are multimeric collagen-like proteins consisting of an N-terminal domain, a collagen-like domain (CD), 2 and a C-terminal fibrinogen-like (FBG) domain involved in innate immune defense (1,2). In humans, three types of ficolins have been identified as follows: Ficolin-1 (M-ficolin), Ficolin-2 (L-ficolin), and Ficolin-3 (H-ficolin/Hakata antigen). They function as recognition molecules in the lectin complement pathway along with mannose-binding lectin but with differentiated complement activating capacity (3). Ficolin-2 and Ficolin-3 circulate in the blood with a median concentration of 5 and 25 g/ml, respectively (4,5). Ficolin-2 is mainly produced in the liver, whereas Ficolin-3 is synthesized in both the liver and lungs, with the highest expression in the lungs (3). Ficolin-1 is primarily expressed by bone marrow-derived cells and lung epithelial cells (3, 6 -8) and has recently been shown to be present in the blood with a median plasma concentration of 60 ng/ml (9). The ficolin genes (FCN1, -2, and -3) are polymorphic, and particularly polymorphisms in FCN2 regulate both the level and function of Ficolin-2 (4,10,11). In this respect, a base substitution in exon 8 at position 6359 (C3 T) causing a threonine to be replaced by a methionine (T236M) in the FBG domain of Ficolin-2 has been shown to cause decreased binding activity toward GlcNAc (10).
Ficolin-1 has been reported to bind to GlcNAc, GalNAc, and sialic acid (8,12). It may opsonize Staphylococcus aureus via GlcNAc and interact with a smooth-type strain of Salmonella typhimurium through an unknown ligand, the binding of which is not diminished by GlcNAc (8). Ficolin-2 has been shown to recognize specific pathogen-associated molecular patterns, 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 walls, and 1,3-␤-D-glucan in yeast and fungal cell walls (13,14). 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 choline (15). Ficolin-2 recognizes clinically important pathogens, like S. typhimurium, S. aureus, and Streptococcus pneumoniae (13,16,17). Ficolin-3 shows affinity for GlcNAc, Gal-NAc, and D-fucose and may interact with S. typhimurium, Salmonella minnesota, and Aerococcus viridans (17,18). The long pentraxin 3 (PTX3) is a soluble pattern recognition molecule mediating innate immune recognition (19). PTX3 is a glycoprotein of 45 kDa, which assembles into an octameric structure through protomer linkage by disulfide bonds (20). PTX3 shares C-terminal structural similarity with the classic short pentraxins, C-reactive protein (CRP), and serum amyloid P component, whereas the N-terminal sequence differs from the other proteins (21). Myeloid cells are a major source of PTX3, but PTX3 has also been shown in vitro to be produced by a variety of cells in response to inflammatory signals (21). During inflammation PTX3 is rapidly up-regulated and released into the surrounding tissue and into the bloodstream. PTX3 interacts with C1q and participates in activation of the classical complement pathway (22,23). Moreover, it has also been shown that PTX3 binds the complement regulatory factor H and that this interaction regulates the alternative pathway of complement (24).
PTX3 can interact with a number of different pathogens, bacteria as well as fungi and viruses. A specific binding has been observed for selected Gram-positive and Gram-negative bacteria, including S. aureus, Pseudomonas aeruginosa, S. typhimurium, Klebsiella pneumoniae, S. pneumoniae, and Neisseria meningitidis (21). PTX3 also binds zymosan and conidia from Aspergillus fumigatus) (25). Furthermore, it has been shown that ptx3 knock-out mice are extremely susceptible to invasive pulmonary aspergillosis. The phenotypic defect can be completely reversed by treatment with recombinant PTX3 (25,26). These data indicate that PTX3 is important in protection against A. fumigatus, which has become a major cause of morbidity in medical institutions because of the increasing number of immunosuppressed patients (27).
Based on the knowledge of the structural and functional similarities between C1q and the ficolins, this study was designed to characterize a possible interaction between the ficolins and PTX3 using A. fumigatus as a model. Based on our data, we propose an important role for previously unlinked collaboration of PTX3 and Ficolin-2, but not Ficolin-1 and Ficolin-3, in the recognition of A. fumigatus and amplification of complement activation. Moreover, our results demonstrate functional consequences of the Ficolin-2 T236M substitution in the interaction between PTX3 and A. fumigatus.
Construction of FCN Expression Plasmids-Liver cDNA was used as a template for PCR amplification for FCN2 and FCN3 and peripheral leukocyte cDNA for FCN1. Oligonucleotide primer sets were designed (FCN-1, sense primer 5Ј-ctagtctagagcgagatggagctgagtggagcca-3Ј and antisense primer 5Ј-accggaattccctaggcgggccgcaccttcat-3Ј; FCN-2, sense primer 5Јctaggtcgacgcgagatggagctggacagagc-3Ј and antisense primer 5Ј-accgcccgggcctaggcaggtcgcaccttcat-3Ј; and FCN-3, sense primer 5Ј-ctaggtcgacgcgagatggatctactgtggatcctg-3Ј and antisense primer 5Ј-accgcccgggcctatcgaagcatcatccgaac-3Ј) to obtain an XbaI site at the 5Ј-end and an EcoRI site at the 3Ј-end for FCN1 and a SalI site at the 5Ј-end and an SmaI site at the 3Ј-end for FCN2 and FCN3, respectively. The amplified products were ligated into the mammalian expression vector pEDdC.
The Ficolin-2 T236M variant was generated using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol, using the FCN2 ligated-pEDdC cDNA described above as a template. One set of oligonucleotides (5Ј-ggagattccctgatgttccacaacaac-3Ј and its reverse complement 5Ј-gttgttgtggaacatcagggaatctcc-3Ј) was designed for generating a point mutation at the amino acid residue at position 236. Constructions were confirmed by Big-Dye terminator cycle DNA sequencing. The final constructions were purified by EndoFree plasmid maxi kit preparation for subsequent transfection.
Transfection and Protein Expression-The pEDdC vector carries a cloning sequence for insertion of the target gene followed by the selectable and amplifiable marker gene dihydrofolate reductase. The expression vectors described above were transfected into the CHO DG44 cell line by using Lipofectamine as described previously (29). Briefly, the untransfected cells were cultured with Iscove's modified Dulbecco's medium supplemented by HT media supplement (1ϫ), 2 mM L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 10% heat-inactivated FBS at 37°C in 5% CO 2 , 95% humidity. Cell splitting was conducted with 0.05% trypsin in PBS. Transfection for obtaining stable cell lines was performed by using the Lipofectamine PLUS reagent kit. Transfection was performed by seeding 8 ϫ 10 5 cells in 6-cm culture wells.
The first selection of stable cell line was performed using G418 to obtain FCN1, FCN2, FCN3, and FCN2-T236M mutant-positive cells, respectively. The second selection and gene amplification were conducted with MTX at a final concentration of 50 nM with G418 supplementation (0.5 mg/ml) and by omitting hypoxanthine and thymidine at the beginning. When the cell viability recovered, 100 and 200 nM of MTX were sequentially applied for the subsequent selection. Afterward, the stable cell line was utilized to produce recombinant Ficolin-1, -2, and -3 and Ficolin-2 mutant by culturing in serum-free medium (PowerCHO-1 CD). The concentrations of ficolins in serum-free medium were calculated by ELISA from a standard curve, which was fitted using previously characterized recombinant Ficolin-1, -2, and -3 with a His tag produced in our laboratory (5,9,29).
SDS-PAGE and Western Blots-Proteins were separated by NuPAGE 3-8% Tris acetate gels or 10% Bistris gels under nonreducing or reducing conditions according to the method of Laemmli (30) and stained with Coomassie Brilliant Blue. The separated proteins were transferred to nitrocellulose (Hybond, ECL, RPN78D, GE Healthcare) using the Xcell II mini-cell blot apparatus in NuPAGE transfer buffer. For detection of Ficolin-1, polyclonal anti-Ficolin-1 antibody was applied as primary antibody. For detection of Ficolin-2 and Ficolin-2 T236M, biotinylated FCN219 was used as primary antibody. Biotinylated polyclonal anti-Ficolin-3 antibody was utilized to detect Ficolin-3. A rabbit polyclonal antibody was used to detect C1q. In the subsequent procedure, blots were stained with HRP-conjugated donkey anti-rabbit IgG antibody, HRP-conjugated rabbit anti-mouse IgG, or HRP-linked streptavidin. Development was performed with SuperSignal West Femto maximum sensitivity substrate on autoradiographic films. Precision prestained protein standard (Bio-Rad) was utilized as a molecular weight standard.
Affinity Chromatography-Human EDTA-plasma (25 ml) from healthy donors was diluted in PBS and applied to CNBr-Sepharose beads (Amersham Biosciences) derivatized with recombinant PTX3. CNBr-Sepharose beads derivatized with BSA (Sigma) and recombinant MBL were used as control. The unbound material was washed from the column with 10-bed column volumes of PBS ϩ 0.05% Tween 20 (Merck). The bound proteins were eluted with 0.1 M glycine-HCl, pH 2.8, immediately adjusted to pH 7.5 with 1.5 M Tris-HCl, pH 8.8, and collected directly into a 5-kDa cut-off Vivaspin centrifuge filter (Sartorius, Epsom, UK). Fractions (5 g of total proteins) were analyzed by SDS-PAGE and Western blot.
In some experiments, plates were coated with PTX3, followed by incubation of Ficolin-1, -2, or -3 in the presence or absence of GlcNAc at concentrations ranging from 0 to 300 mM, and also with 5 mM of the calcium chelator EGTA. Bound ficolins were detected as depicted above.
To compare natural ficolins and recombinant ficolins, plates were coated with recombinant PTX3, followed by addition of natural and recombinant Ficolin-2 or -3, respectively. Bound ficolins were detected as described above. Alternatively, wells coated with natural PTX3 or recombinant PTX3 were incubated with recombinant Ficolin-1, -2, and -3, respectively.
Binding of Ficolin-2 to PTX3 Using Surface Plasmon Resonance Spectroscopy-Analyses were performed using a BIAcore 3000 instrument (GE Healthcare). Recombinant PTX3 was diluted to 100 g/ml in 10 mM sodium formate, pH 3.5, and immobilized on the surface of a CM5 sensor chip (GE Healthcare) using the amine coupling chemistry. Binding of recombinant purified Ficolin-2 (29) to immobilized PTX3 (4000 resonance units) was measured at a flow rate of 20 l/min in 150 mM NaCl, 2 mM CaCl 2 , 20 mM HEPES, pH 7.4, containing 0.005% Ficolins and PTX3 OCTOBER 9, 2009 • VOLUME 284 • NUMBER 41 surfactant P20 (GE Healthcare). Regeneration of the surface was achieved by injection of 10 l of 1 M sodium acetate, pH 7.2 followed by 10 l of 10 mM NaOH. Equivalent volumes of each protein sample were injected over an activated-deactivated surface to serve as blank sensorgrams for subtraction of the bulk refractive index background.
Data were analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases for six concentrations simultaneously, using the BIAevaluation 3.2 software (GE Healthcare). The apparent equilibrium dissociation constants (K D ) were calculated from the ratio of the dissociation and association rate constants (k off /k on ). The molar concentration of Ficolin-2 was estimated assuming a tetrameric structure of 414 kDa (12 polypeptide chains of 34.5 kDa each) (29).
Binding of Wild Type Ficolin-2 and Its Mutant to GlcNAc Beads-Three g of wild type Ficolin-2 or Ficolin-2 T236M were incubated with 50 l of GlcNAc-agarose beads overnight with continuous stirring at 4°C. Total reaction volume was made up to 1.5 ml with TBS-Ca 2ϩ as binding buffer. Following centrifugation at 1000 rpm for 1 min, the beads were washed three times with binding buffer. Bound proteins were eluted with 0.4 M GlcNAc in binding buffer. The concentration of wild type Ficolin-2 and Ficolin-2 T236M in eluates was measured by ELISA as described above.
PTX3 Binding to A. fumigatus-To verify PTX3 binding to A. fumigatus, PTX3 was incubated with A. fumigatus at a final concentration of 10 g/ml in the absence or presence of 20 g/ml galactomannan at 37°C for 1 h, followed by incubation of biotinylated anti-PTX3 monoclonal antibody at 4°C for 30 min. PTX3 binding was detected by incubation of streptavidin/phycoerythrin at 4°C for 15 min and measured by FACS analysis.
Interaction between Ficolin-2 and PTX3 on A. fumigatus-The procedure of Ficolin-2 binding to A. fumigatus was the same as mentioned above. A. fumigatus was incubated with Ficolin-2 (7 g/ml), followed by incubation with PTX3 (10 g/ml) at 37°C for 1 h. Bound PTX3 was detected as indicated above. Alternatively, PTX3 was incubated with A. fumigatus at 37°C for 1 h prior to incubation with Ficolin-2. Binding of Ficolin-2 in the presence of PTX3 was also measured using the same method.
In some experiments, A. fumigatus conidia were incubated with Ficolin-2 T236M instead of wild type Ficolin-2. Ficolin-2 T236M binding was detected with monoclonal anti-Ficolin-2 antibody FCN219 by incubation at 4°C for 30 min and measured by FACS analysis.
C4 Deposition on A. fumigatus-C4 deposition on A. fumigatus was assessed by FACS analysis. As source of complement component, C1q-deficient serum was used in subsequent experiments. C1q-deficient serum was obtained from a C1qdeficient individual with normal levels of Ficolin-2 and MBL (31). Subsequently, MBL was depleted by agitating the C1qdeficient serum with mannan-agarose beads at 4°C for 2 h. To deplete serum Ficolin-2, magnetic pan-mouse IgG Dynabeads was applied according to the manufacturer's instructions. In brief, Ficolin-2 was depleted by incubation of the MBL-depleted and C1q-deficient serum with FCN219 (10 g/ml) and Dynabeads at 4°C with rotation. 20% C1q-deficient serum depleted of MBL (C1q Ϫ /MBL depleted serum) and Ficolin-2 (C1q Ϫ /MBL, Ficolin-2 depleted serum) was prepared by dilution of the C1q Ϫ /MBL depleted serum in HEPES buffer.
For C4 deposition, A. fumigatus conidia were incubated with 20% C1q Ϫ /MBL depleted serum with supplementary C4 (10 g/ml) at 37°C for 1 h. Alternatively, 20% C1q Ϫ /MBL, Ficolin-2 depleted serum reconstituted by Ficolin-2 (7 g/ml) was incubated with A. fumigatus conidia with supplementary C4 (10 g/ml) at 37°C for 1 h. All reaction volumes were 100 l, and A. fumigatus was washed after each step in HEPES buffer containing 1% heat-inactivated FCS. C4 deposition was detected with a rabbit anti-human C4 antibody by incubation at 4°C for 30 min, followed by incubation with swine anti-rabbit IgG FITC conjugate at 4°C for 15 min. Finally, C4 deposition was assessed by FACS analysis, and data were analyzed by CellQuest Pro software (BD Biosciences).
In some experiments, A. fumigatus conidia were preincubated with 10 g/ml PTX3 at 37°C for 1 h prior to assessment of C4 deposition. As a control, MBL levels in C1q-deficient serum and C1q Ϫ /MBL depleted serum were determined by assessing MBL binding to A. fumigatus. A. fumigatus conidia were incubated with 20% C1q Ϫ /MBL depleted serum at 37°C for 1 h. Bound MBL was detected with anti-MBL monoclonal antibody (HYB 131-11) and goat anti-mouse IgG FITC conjugate by incubation at 4°C for 30 and 15 min, respectively. Furthermore, Ficolin-2 levels in C1q Ϫ /MBL, Ficolin-2 depleted serum were determined by Western blot. C1q Ϫ /MBL depleted serum before and after depletion (5 g of total proteins) was analyzed by SDS-PAGE under reducing conditions, followed by detection of biotinylated rabbit polyclonal anti-Ficolin-2 antibody and streptavidin-HRP, respectively. As a supplementary experiment, recombinant Ficolin-2 (7.0 g/ml) and recombinant MASP-2 (1.0 g/ml) were applied as substitute complement initiators instead of serum.
Statistical Analysis-Data represent the means Ϯ S.D. or Ϯ S.E. of at least three independent experiments. The means Ϯ S.D. were calculated by Excel software (Microsoft). Student's t test was used to calculate significance, and p Ͻ 0.05 was considered to represent a statistical significant difference between two sample means.

Ficolin-2 Isolation from Human Plasma by Sepharose-PTX3
Beads-In ligand fishing experiments of human plasma involving PTX3 affinity chromatography coupled to mass spectrometry, 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 Sepharose-PTX3 beads incubated with human plasma. In Fig. 1, representative experiments with Ficolin-2 and C1q are shown. Sepharose-BSA and MBL beads were used as controls.
Recombinant Ficolin-1, -2, and -3 Characterization-Recombinant 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 containing a dihydrofolate reductase expression site for MTX-induced amplification. CHO DG44 cell lines were transfected with the expression vectors containing the FCN1, -2, and -3 insert along with pSV 2neo containing a neomycin resistance site and selected with G418. Clones expressing ficolins from G418 selection were generated through multicycle of amplification with elevated concentration of MTX. Using ELISA, the concentrations of Ficolin-1, -2, and -3 in the culture supernatants were quantified as 12, 7, and 10 g/ml, respectively. To determine the oligomerization state of ficolins, recombinant and natural Ficolin-1, -2, and -3 were analyzed by 3-8% Tris acetate SDS-PAGE under nonreducing or reducing conditions, followed by Western blot (Fig. 2). Under nonreducing conditions, Ficolin-1, -2, and -3 presented multimeric assembly laddered with Ͼ10 bands (lane 1 in Fig. 2, A-C) but ran as a single band of ϳ34, 35, and 34 kDa under reducing conditions on Western blot, respectively (lane 2 in Fig. 2, A-C). The multimeric pattern is in correspondence with the pattern seen in a serum pool (Fig. 2, D-F). This result showed that CHO cells secreted recombinant Ficolin-1, -2, and -3 were oligomerized in subunits stabilized via

Ficolins and PTX3
OCTOBER 9, 2009 • VOLUME 284 • NUMBER 41 disulfide bonds, ranging from two to more than four subunits in a similar fashion to natural proteins.
Ficolin-1 exhibited better binding to PTX3 than Ficolin-3. To further determine the binding specificity of Ficolin-1, -2, and -3, we next examined their binding to immobilized PTX3 in the presence of GlcNAc (Fig. 4, A-C). Inclusion of GlcNAc in the binding buffer attenuated the binding of Ficolin-1 and -2 to immobilized PTX3, although no difference in Ficolin-3 binding was detected in the presence or absence of GlcNAc. These results implied that Ficolin-2, in particular, but also Ficolin-1 bind to immobilized PTX3 via the FBG domain but not Fico-

Ficolins and PTX3
lin-3. We then removed calcium by adding a buffer containing 5 mM EGTA in experiments, including Ficolin-1 and Ficolin-2 (Fig. 5, A and B). These experiments resulted in a substantial increase in binding of both Ficolin-1 and Ficolin-2 to PTX3, but GlcNAc could still inhibit the interactions.
The ability of recombinant Ficolin-2 to interact with PTX3 was also investigated by surface plasmon resonance spectroscopy. As shown in Fig. 6, Ficolin-2 bound to immobilized PTX3 in the presence of 2 mM CaCl 2 . The kinetic parameters of the interaction were determined by recording sensorgrams at varying ficolin concentrations (Fig. 6A) and evaluating the data by global fitting as described under "Experimental Procedures." A k on value of 6.0 ϫ 10 5 M Ϫ1 s Ϫ1 and a k off value of 3.9 ϫ 10 Ϫ3 s Ϫ1 were determined, yielding a resulting apparent K D value of 6.5 nM, indicative of a high affinity interaction. Binding was maintained when EDTA was substituted for Ca 2ϩ in the running buffer, and the complex appeared even more stable, as reflected by the slower dissociation phase (Fig. 6B).
Relative Binding of Natural and Recombinant Ficolins to PTX3-To determine binding capacity of recombinant ficolins to PTX3 compared with ficolins from natural material, ELISA wells coated with recombinant PTX3 were incubated with each natural or recombinant ficolins. Both natural and recombinant Ficolin-2 showed vigorous binding to recombinant PTX3 in a similar manner (Fig. 7A). However, natural Ficolin-3 presented no binding compared with slight binding of recombinant Ficolin-3 (Fig. 7A). Subsequently, we also evaluated binding of recombinant ficolins to natural and recombinant PTX3. ELISA wells coated with natural or recombinant PTX3 were incubated with each recombinant ficolin. Recombinant Ficolin-2 bound both natural and recombinant PTX3; however, the binding to natural PTX3 is dominant, approximately three times higher than to recombinant PTX3 (Fig. 7B). Furthermore, recombinant Ficolin-1 and -3 presented very low binding to both natural and recombinant PTX3 (Fig. 7B). No signal was detected in BSA-coated wells (Fig. 7).
Binding of Ficolin-2 to A. fumigatus-To further provide evidence that the observed interaction between ficolins and PTX3 may be physiologically relevant, we focused our attention on A. fumigatus as an anchored matrix. To assess interaction of the ficolins with PTX3 on A. fumigatus, we first examined whether ficolins by themselves were able to bind to the surface of A. fumigatus by FACS analysis.
As shown in Fig. 8, A and B, we found that Ficolin-2 did bind to A. fumigatus directly in a dose-dependent manner, but neither Ficolin-1 nor -3 did. As a control, binding of recombinant MBL was also assessed concurrently. The binding of Ficolin-2   OCTOBER 9, 2009 • VOLUME 284 • NUMBER 41 to A. fumigatus was significantly inhibited in the presence of 0.3 M GlcNAc (Fig. 8C). Excess mannose or EDTA had no significant effect on Ficolin-2 binding to A. fumigatus (Fig. 8C). In contrast, binding of MBL to A. fumigatus was drastically decreased in the presence of GlcNAc or mannose or EDTA (data not shown).
Collaboration of Ficolin-2 and PTX3 on A. fumigatus-To address whether an interaction between Ficolin-2 and PTX3 may occur on the surface of A. fumigatus, we assessed binding by FACS analysis and compared the reactions in the presence of either PTX3 or Ficolin-2 alone or together (Fig. 9, A-C). As shown in Fig. 9, B and C, Ficolin-2 bound to A. fumigatus much better than PTX3, but most interestingly preincubation of PTX3 increased Ficolin-2 binding to A. fumigatus significantly (p Ͻ 0.05) (Fig. 9B) and vice versa (p Ͻ 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 Ϫ / MBL depleted serum. MBL depletion was assessed by analysis of MBL binding to A. fumigatus, compared with control reaction without MBL (Fig. 10A). Furthermore, binding of serum Ficolin-2 to A. fumigatus was also confirmed using C1q Ϫ / MBL depleted serum (Fig. 10B) before testing C4 deposition, 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 Ϫ /MBL depleted serum verified above. In C1q Ϫ /MBL depleted serum we could observe deposition of C4 in the absence of PTX3 (Fig. 10C). However, this deposition was significantly increased in the presence of PTX3 (p Ͻ 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 Ϫ /MBL depleted serum and assessed by Western blot as 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 Ϫ / MBL, Ficolin-2 depleted serum was reconstituted by addition of Ficolin-2. Consistently, this deposition was significantly increased in the presence of PTX3 (p Ͻ 0.05), compared with control experiment without PTX3 (Fig. 10D).
To further provide evidence for the preceding results, C1q Ϫ / MBL depleted serum was replaced with a more simple system, recombinant Ficolin-2 and MASP-2 as substitute complement initiators in the presence of exogenous C4. As shown in Fig.  10E, additional Ficolin-2-MASP-2 complex leads to C4 deposition on A. fumigatus. Consistent with the above results, PTX3 significantly enhanced Ficolin-2-MASP-2 complex-induced C4 deposition on A. fumigatus (p Ͻ 0.05), compared with control experiment without PTX3 (Fig. 10E).
Reduced Ficolin-2 T236M Binding to GlcNAc, PTX3, and A. fumigatus-Previously we found an allelic variant in the FCN2 gene that replaced a threonine with a methionine at amino acid position 236 (Ficolin-2 T236M), which when purified from serum exhibited reduced binding capacity to GlcNAc compared with wild type Ficolin-2 (10). To clarify whether recombinant Ficolin-2 T236M also exhibited altered or decreased ligand-binding capacity, an expression vector coding for the Ficolin-2 T236M was generated by site-directed mutagenesis, and the mutated protein was expressed in CHO cells. SDS-PAGE followed by Western blot analysis showed that the protein had a multimeric pattern comparable with that of the wild type protein (Fig. 11A). Moreover, binding of Ficolin-2 T236M to previously substantiated ligands GlcNAc, PTX3, and A. fumigatus was assessed by ELISA and FACS analysis, respectively, as described under "Experimental Procedures." Differential binding capacity was detected between wild type Ficolin-2 and its T236M mutant. In accordance with our previous data, recombinant Ficolin-2 T236M showed signif- 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 recombinant Ficolin-2 and -3 or serum Ficolin-2 and -3 (5 g/ml). Bound Ficolin-2 and -3 were detected. B, microtiter wells coated with recombinant PTX3, 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 means Ϯ S.D. from triplicate experiments. Results are representative from two independent experiments that yield similar results.

DISCUSSION
It is well described that C1q interacts with the short pentraxin family members CRP and serum amyloid P component and that this complex formation may mediate classical pathway complement activation (33,34). The same has been shown for solid phase bound PTX3 (22,23). This complex triggers activation of the classical pathway of complement via the globular heads of C1q.
Whether the lectin pathway initiators may have similar functions is largely unknown. However, it has recently been shown that both Ficolin-1 and Ficolin-2 interact with CRP and that this complex may mediate complement killing of P. aeruginosa (35,36). We have demonstrated that circulating plasma-derived Ficolin-2 interacted with immobilized PTX3 in affinity chromatography experiments. This is similar to what was previously shown in the case of C1q in binding experiments (22). Moreover, Ficolin-2 and Ficolin-1 were able to bind to PTX3 adsorbed to microtiter wells in a dose-dependent fashion. These interactions were partially inhibited by GlcNAc. We also observed some binding with Ficolin-3, but we were not able to inhibit the binding with GlcNAc and interpret this result as a nonspecific interaction. Zhang et al. (36) showed that at pH 7.4 an increased calcium concentration dramatically inhibited the CRP/Ficolin-2 interaction, indicating that calcium under normal conditions prevents the CRP/Ficolin-2 interaction. Our  OCTOBER 9, 2009 • VOLUME 284 • NUMBER 41 experiments were usually performed at pH 7.4 at a calcium concentration of 5 mM showing that the interaction between PTX3 and Ficolin-1 and in particular Ficolin-2 could be oper-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 plasmon 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 pattern as in the presence of 5 mM calcium (data not shown). Thus, we choose to continue the experiments using calcium-containing buffers, which are closer to physiological conditions. However, according to our results using natural and recombinant proteins, it should be noted that natural Ficolin-2 and recombinant 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 recombinant 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 vigorous interaction in vivo. Nevertheless, because of very low levels 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 composition of recombinant and natural PTX3, indicating no significant differences between PTX3 from natural material or CHO expression system.

Ficolins and PTX3
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 cytometry 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 was reduced to ϳ50% in the presence of GlcNAc. The latter finding suggests that more than one binding site in the FBG domain of Ficolin-2 is involved in the interaction with A. fumigatus. Recently, the crystal structure of the FBG domain of ficolins has been solved and has revealed the location of the major ligand-binding sites of Ficolin-2 (39). MBL has only one specific binding site in the CRD domain to recognize C3-OH and C4-OH of terminal carbohydrate residues by a typical hydrogen bonding interaction (40). In addition to a common external S1-binding site that it shares with Ficolin-1 and Ficolin-3, Ficolin-2 has three additional binding sites (S2-S4) in the FBG domain (39). These findings explain why Ficolin-2 harbors the capacity of versatile recognition of a variety of acetylated and carbohydrate-containing compounds. Most of the acetylated ligands bind to sites S2 and S3 of the FBG domain of Ficolin-2, whereas the binding of ␤-1,3-glucan involves the edge of site S3 and S4 via water-mediated hydrogen bonding interactions. However, the S3-binding site is shared by both the acetyl group of GlcNAc and the first glucose residue of an elongated ␤-1,3-glucan molecule (39). Because the cell membrane of A. fumigatus is composed mainly of chitin (a polymer of ␤-1, 4-N-acetyl-glucosamine), ␤-1,3-glucan, and galactomannan (41), it is reasonable to hypothesize that two kinds of recognition sites are involved in the binding of Ficolin-2 to A. fumigatus, which may both include a GlcNAc and a ␤-1,3-glucan moiety. Our competition experiments using Curdlan (a ␤-1,3-Glucan hydrate from A. faecalis) suggest that the FBG domain of Ficolin-2 preferentially recognizes the ␤-1,3-glucan moiety in addition to the GlcNAc moiety on A. fumigatus, which accounts for the strong combined inhibiting effect of the two compounds. In agreement with the fact that the ligand-binding sites are distant from the Ca 2ϩ -binding site in the Ficolin-2 structure, A. fumigatus recognition is not sensitive to EDTA.
PTX3 clearly bound A. fumigatus as has been shown before (25,26), but to a lesser degree than Ficolin-2. Of particular interest was the observation that PTX3 could significantly augment the deposition of Ficolin-2 on the surface of A. fumigatus. However, a possibly more intriguing finding was the observation that Ficolin-2 by itself could enhance the binding of PTX3 to A. fumigatus. This indicates that PTX3 also interacts with Ficolin-2 at sites that are not involved in Ficolin-2 interaction with A. fumigatus per se. This may also explain the partial inhibition of the interaction between Ficolin-2 and PTX3 that we observed with GlcNAc on the solid phase polystyrene matrix. To further elucidate whether the Ficolin-2/PTX3 interactions with A. fumigatus may be of physiological relevance, we investigated their influence on complement activation induced by Ficolin-2. We could show that Ficolin-2 and PTX3 collaborate to boost Ficolin-2-mediated complement deposition. Thus, it is tempting to speculate that this interaction may play a significant physiological role. Ficolins and PTX3 OCTOBER 9, 2009 • VOLUME 284 • NUMBER 41 No deficiency state of Ficolin-2 in humans has been described so far, but promoter variants in the Ficolin-2 gene (FCN2) are associated with differences in the serum concentrations (4,10). However, the GlcNAc binding ability of Ficolin-2 is hampered by a polymorphism situated in exon 8 in the FCN2 gene causing a threonine to be replaced with methionine (T236M) (10). This observation was based on binding studies performed with Ficolin-2 in whole serum. To bring the understanding of this observation a step further, we produced the variant in a recombinant form as we have done with wild type Ficolin-2. The T236M variant folded correctly into high order oligomeric structures comparable with those obtained for wild type Ficolin-2, as judged from SDS-PAGE and Western blot analysis. As we have shown for serum Ficolin-2 T236M variant (10), the GlcNAc binding capacity of the recombinant variant was dramatically decreased when compared with the wild type protein as was the interaction with PTX3 and A. fumigatus. Thus, this finding demonstrates the existence of a functional Ficolin-2 deficiency that may be of pathophysiological relevance not only in the direct interaction between Ficolin-2 and 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 crystallographic observations.
In summary, plasma Ficolin-2 could be purified with immobilized PTX3. Ficolin-1 and in particular Ficolin-2 but not Ficolin-3 interacted with PTX3 in a calcium-independent manner. Only Ficolin-2 bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-mediated complement deposition on the surface of A. fumigatus was enhanced by PTX3. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens and enhance complement activation. This effect was dramatically reduced by a common amino acid change in the FBG domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.