L-ficolin Is a Pattern Recognition Molecule Specific for Acetyl Groups*

L-ficolin and H-ficolin are molecules of the innate immune system. Upon recognition of a suitable target they activate the complement system. The ligand recognition structure of ficolins is contained within a fibrinogen-like domain. We examined the selectivity of the ficolins through inhibiting the binding to bacteria or to beads coupled with N -acetylglucosamine. The binding of L-ficolin to Streptococcus pneumoniae 11F and the beads was inhibited by N -acetylated sugars and not by non-acetylated sugars. However, it was also inhibited by other acetylated compounds. Based on this selectivity L-ficolin is not easily defined as a lectin. The binding of H-ficolin to Aerococcus viridans was not inhibited by any of the sugars or other compounds examined. Based on the selectivity of L-ficolin we developed a new purification procedure involving affinity chromatography on N -acetylcysteine-deriva-tized Sepharose. The column was loaded in the presence of EDTA and high salt, and L-ficolin was eluted by decreasing the salt concentration. Further purification was achieved by ion exchange chromatography. The immune system is comprised of innate and adaptive defense mechanisms. The innate immune system prevents or limits the early stages of an infection and is further involved in the orchestration of the ensuing adaptive immune response (1). different recognition and effector mech-anisms the derivatized sulfone incubation with 10% (v/v) divinyl sulfone (Sigma-Aldrich) in 0.5 M Na 2 CO 3 , pH 11, for 1.5 h at room temperature. were washed with 0.25 M Na 2 CO 3 , pH 11, and derivatized in 0.25 M Na 2 CO 3 containing 10% (w/v) GlcNAc or CysNAc at pH 11 at were H O, residual were blocked by incubation M 9.0. derivatized

L-ficolin and H-ficolin are molecules of the innate immune system. Upon recognition of a suitable target they activate the complement system. The ligand recognition structure of ficolins is contained within a fibrinogen-like domain. We examined the selectivity of the ficolins through inhibiting the binding to bacteria or to beads coupled with N-acetylglucosamine. The binding of L-ficolin to Streptococcus pneumoniae 11F and the beads was inhibited by N-acetylated sugars and not by non-acetylated sugars. However, it was also inhibited by other acetylated compounds. Based on this selectivity L-ficolin is not easily defined as a lectin. The binding of H-ficolin to Aerococcus viridans was not inhibited by any of the sugars or other compounds examined. Based on the selectivity of L-ficolin we developed a new purification procedure involving affinity chromatography on N-acetylcysteine-derivatized Sepharose. The column was loaded in the presence of EDTA and high salt, and L-ficolin was eluted by decreasing the salt concentration. Further purification was achieved by ion exchange chromatography.
The immune system is comprised of innate and adaptive defense mechanisms. The innate immune system prevents or limits the early stages of an infection and is further involved in the orchestration of the ensuing adaptive immune response (1). It encompasses many different recognition and effector mechanisms including the complement system. The complement system can be activated through three different pathways; they are the classical, the alternative, and the mannan binding lectin (MBL) 1 pathways. All three pathways merge in the formation of a C3 convertase, C4bC2b for the classical and the MBL pathway and C3bBb for the alternative pathway. MBL circulates in complex with four different proteins, three MBLassociated serine proteases (MASPs) (MASP-1, -2, -3) and a smaller protein MAp19 or sMAP (2)(3)(4). MASP-2 has been identified as the protease responsible for generating the C3 convertase through cleavage of C4 and C2 (3).
Recently a group of proteins, the ficolins, which structurally resemble MBL, has been discovered. Three members of the ficolin family have been identified in humans: L-ficolin, Mficolin, and H-ficolin. L-ficolin and H-ficolin are plasma proteins of hepatic origin present in concentrations ranging from 1 to 14 and 7 to 23 g/ml, respectively (5)(6)(7). H-ficolin is also found in secretions, i.e. in alveoli and in bile (8). M-ficolin is produced by non-differentiated monocytes and has so far been found only on the surface of these cells (9). The polypeptide chains of the ficolins are, like those of MBL, comprised of different structural regions, an N-terminal region, a collagenlike region, and a globular domain (10). Compared with MBL the main structural difference is the lack of an ␣-helical region and a globular domain which is a fibrinogen-like (fbg) domain rather than the C-type lectin domain of MBL (11). Like MBL, L-ficolin and H-ficolin are capable of forming trimeric subunits, which associate into oligomers comprised of up to 4 and 6 trimers, respectively (12,13). Like MBL, L-ficolin and H-ficolin circulate in complex with MASPs and are capable of activating the complement system (14 -16). Because of the structural, biochemical, and functional similarities to MBL, the ficolins are also believed to be involved in the innate immune defense as pattern recognition molecules, but so far little is known about their possible antimicrobial properties. L-ficolin has been demonstrated to activate complement upon binding to Salmonella typhimurium and lipoteichoic acids (14,17). H-ficolin has been shown to activate complement (16) and to inhibit the growth of an Aerococcus viridans strain (7).
The ligands for the ficolins have been suggested to be monosaccharides (18,19), and the fbg domain of tachylectin 5A, an invertebrate analogue, has been crystallized in complex with N-acetylglucosamine (GlcNAc) (20). We now report further investigations of the specificity of the human ficolins partly conflicting with published results. Based on the knowledge acquired we developed a new method for the purification of L-ficolin.

EXPERIMENTAL PROCEDURES
Glucose (Glc), glucosamine (GlcN), GlcNAc, mannose (Man), mannosamine (ManN), N-acetylmannosamine, galactose (Gal), galactosamine (GalN), N-acetylgalactosamine (GalNAc), glycine, N-acetylglycine, N-acetylcysteine (CysNAc), acetylsalicylic acid, and acetylcholine were purchased from Sigma-Aldrich. A 300 mM stock solution of these compounds was made in 20 mM Tris-HCl, 140 mM NaCl, 1.5 mM NaN 3 , pH 7.4 (TBS) with 0.05% (v/v) Tween 20 and 5 mM CaCl 2 (TBS/Tw/Ca). The pH was adjusted with 5 N NaOH to pH 7.4 if needed. Sepharose 4B CL beads (Amersham Biosciences) were derivatized with GlcNAc and CysNAc. The beads were activated with divinyl sulfone by incubation with 10% (v/v) divinyl sulfone (Sigma-Aldrich) in 0.5 M Na 2 CO 3 , pH 11, for 1.5 h at room temperature. The beads were washed with 0.25 M Na 2 CO 3 , pH 11, and derivatized in 0.25 M Na 2 CO 3 containing 10% (w/v) GlcNAc or CysNAc at pH 11 overnight at room temperature. The beads were washed with H 2 O, and residual active groups were blocked by incubation with 0.1 M ethanolamine, pH 9.0. The derivatized beads were washed with TBS and stored at 4°C. We also used bacteria as ligands. The bacteria were grown in Todd-Hewitt broth medium (Oxoid, Basingstoke, Great Britain) at 5% CO 2 or in L-Broth medium (Qbiogene, Carlsbad, CA) overnight at 37°C for Streptococcus pneumoniae 11F (Statens Serum Institut, Copenhagen, Denmark) and A. viridans 86965 (21), respectively. Formaldehyde (Sigma-Aldrich) was added to the broth cultures to a final concentration of 1% (w/v) and kept at room temperature until the next day. Residual reactive aldehyde groups were blocked by incubating for 1 h with 0.1 volumes of 1 M ethanolamine, pH 9.0, and the bacteria were washed three times with TBS and stored at 4°C.
Ficolins were purified as described (14). Antibodies used for Western blotting were monoclonal mouse antibody 1E2 directed against the A chain of MASP-1 (since the A chain is shared by MASP-1 and MASP-3, this is an anti-MASP-1/3 antibody) (22), monoclonal rat antibody 6G12 raised against MAp19 (since the domains of MAp19 and the first two domains of MASP-2 are identical, this is an anti-MAp19/MASP-2 antibody) (23), and polyclonal rabbit-anti-L-ficolin antibody (16). Monoclonal anti-L-ficolin antibody (2F5) (6) was biotinylated with 167 g of biotinyl-N-hydroxysuccinimide (Sigma-Aldrich) per mg of protein. The monoclonal anti-H-ficolin antibody (19) (4H5, HyCult Biotechnology, PB Uden, The Netherlands) was biotinylated with 33 g of biotinyl-Nhydroxysuccinimide per mg of protein since loss of activity was observed at a higher degree of biotinylation.
Inhibition Assay-GlcNAc beads (13-l packed volume) or 4.5 ϫ 10 8 A. viridans or S. pneumoniae was mixed with 6 l of serum (containing 5 g of L-ficolin and 20 g of H-ficolin/ml) and further mixed with the inhibitors to give final inhibitor concentrations of 100, 50, 25, 12.5, 3, and 1 mM. The volume was adjusted to 300 l with TBS/Tw/Ca, and the samples were incubated for 2 h at room temperature and centrifuged (100 ϫ g for beads and 10,000 ϫ g for bacteria, 5 min). The supernatants were dialyzed using Slide-A-Lyzer MINI Dialysis units (Pierce) against TBS/Tw/Ca overnight at room temperature, and the amount of L-ficolin and H-ficolin was estimated by the assays described below. Binding to N-Acetylcysteine-derivatized Beads-The beads, produced as described above, were incubated in buffer with an equal volume of a 4 -8% (w/v) polyethylene glycol 6000 (PEG) (Fluka, Buchs SG, Switzerland) cut from serum (described under "Purification of L-ficolin"). The buffer contained 10 mM Tris-HCl, 0.05% (v/v) Tween 20, 2-fold dilutions of NaCl starting at 1 M, and 2 mM CaCl 2 or 5 mM EDTA. The samples were incubated overnight at 4°C and centrifuged (100 ϫ g, 5 min), and the amount of L-ficolin in the supernatant was quantified.
Purification of L-ficolin-Citrated donor plasma was coagulated by the addition of 1 M CaCl 2 to a final concentration of 5 mM and incubated at 37°C for 1 h, and the serum was collected. To establish the concentration of PEG needed for the precipitation of L-ficolin, PEG was dissolved in TBS/Tw/Ca to concentrations of 4, 8, 12, 16, and 20% (w/v), and equal volumes of serum and PEG solutions were mixed, incubated for 30 min, and centrifuged (1000 ϫ g, 5 min). The amounts of MBL (24), C1q (25), L-ficolin, and H-ficolin in the supernatants were quantified. Based on the results, the following procedure for initial purification of L-ficolin was adopted; a solution of 24% (w/v) PEG was added to 900 ml of serum to a final concentration of 4% (w/v) PEG. After incubation for 30 min at room temperature the mixture was centrifuged (1000 ϫ g, 5 min), and the supernatant was collected. PEG (24% (w/v)) was added to the supernatant to obtain a final concentration of 8% (w/v). The mixture was incubated and centrifuged as before. The supernatant was discarded, and the pellet (the 4 -8% PEG cut) was dissolved in 200 ml of 500 mM NaCl, 1.5 mM NaN 3 , 2 mM EDTA, 0.01% (v/v) polyoxyethylene 10 tridecyl ether (Emulfogen, Sigma-Aldrich) (loading buffer) and loaded onto an 8-ml column of N-acetylcysteine beads at 0.5 ml/min. After washing with loading buffer until base-line absorbance was reached, bound L-ficolin was eluted with 10 mM Tris-HCl, 20 mM NaCl, 1.5 mM NaN 3 , 2 mM EDTA, 0.01% (v/v) Emulfogen. The amount of L-ficolin was quantified, and the fractions with the highest contents were pooled and passed onto a 1-ml Mono Q column (Amersham Biosciences). The bound material was eluted by a 20-ml NaCl gradient from 50 to 500 mM, and the fractions containing L-ficolin were identified and pooled.
SDS-PAGE and Western Blotting-Samples were analyzed by SDS-PAGE (4 -20% acrylamide gradient) under reducing and non-reducing conditions followed by silver staining of the proteins (26), or the proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). The blots were probed with the antibodies described above followed by horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and subsequently incubated with enhanced chemiluminescence (ECL) solution (12.5 mM luminol, 0.01% (v/v) H 2 O 2 , 100 mM Tris-HCl, pH 8.5).
Gel Permeation Chromatography-To compare the size of L-ficolin found in serum and the purified preparation, 50 l of serum or 20 g of purified L-ficolin was fractionated on a Superose 6 column (10 mm Sequence Alignment-The primary sequence alignment in this study was performed with ClustalW (www.ebi.ac.uk/clustalw, European Bioinformatics Institute, Heidelberg, Germany). The accession numbers in the NCBI data base used for the primary sequences were for tachylectin 5A (1JC9A), L-ficolin (NP_004099), M-ficolin (O00602), and for H-ficolin (NP_003656).

RESULTS
GlcNAc and A. viridans have previously been described as ligands for L-ficolin and H-ficolin, respectively. We examined the inhibitory potential of various compounds toward L-ficolin and H-ficolin binding, and clear differences were seen. Fig. 1 shows the amount of L-ficolin and H-ficolin bound to GlcNAcbeads and A. viridans, respectively, in the presence of different inhibitors. Fig. 1 shows examples of inhibition curves, whereas Table I summarizes all inhibition data. GlcNAc and N-acetylmannosamine seem to be the most potent inhibitors of L-ficolin, whereas GalNAc, CysNAc, N-acetylglycine, and acetylcholine are about half as potent. The results suggest that acetylated compounds in general inhibit L-ficolin. When the experiments were repeated with S. pneumoniae 11F (previously described as ligand for L-ficolin) instead of GlcNAc beads, it was again found that acetylated carbohydrates inhibit the binding (I 50 for Glc-NAc, N-acetylmannosamine, and GalNAc was determined to 27, 31, and 24 mM, respectively), whereas non-acetylated do not. In the case of H-ficolin none of the compounds tested showed inhibitory activity.
Based on the results obtained by the inhibition experiments, we developed a purification procedure for L-ficolin. Sepharose 4B was derivatized with acetylated compounds. When the potential of the beads for binding L-ficolin were compared, Cys-NAc beads were found to have more than twice the capacity of GlcNAc beads (results not shown). Because MBL is known also to bind to GlcNAc beads, we chose to use CysNAc beads for the purification of L-ficolin. As a first step L-ficolin was precipitated from serum by adding PEG. Fig. 2 shows the amount of L-ficolin, H-ficolin, MBL, and C1q that remained in the supernatant after precipitation at increasing concentrations of PEG. Because ϳ80% of L-ficolin remains in the supernatant after 4% PEG while the amount of both MBL and C1q was reduced dramatically, we decided to make a 4 -8% PEG cut for the purification of L-ficolin.
The ability of L-ficolin in the 4 -8% PEG cut to bind to CysNAc beads was examined. Fig. 3 shows the amount of L-ficolin bound at increasing salt concentrations in the presence or absence of calcium. When calcium was present all the L-ficolin was bound independent of the NaCl concentration.
This binding was partially inhibitable by GlcNAc (not shown).
In the presence of EDTA the binding was markedly dependent of the NaCl concentration. When incubating with EDTA in the presence of 1 or 0.5 M NaCl all the L-ficolin was bound, whereas much less was bound at lower NaCl concentrations. For the following larger scale purification we decided to allow L-ficolin to bind to the CysNAc beads in a buffer with 0.5 M NaCl and 2 mM EDTA followed by elution at low ionic strength (20 mM NaCl, 2 mM EDTA). Fig. 4A shows the loading and elution profile when applying the 4 -8% PEG cut from 900 ml of serum onto the 8 ml of CysNAc column. Clearly the capacity of the column was not exceeded. L-ficolin was eluted after changing the high salt buffer to low salt buffer. After eluting with 20 mM NaCl the L-ficolin-containing fractions were pooled, and the proteins were further fractionated on a Mono Q ion exchange column. L-ficolin was found in two peaks (Fig. 4B), one smaller at ϳ80 mM of NaCl and a major eluting between 150 and 200 mM salt. Analysis by SDS-PAGE revealed no difference between the L-ficolin composition in the two peaks, except from contaminations with unidentified protein in peak 1(not shown). Matsushita et al. (10) found L-ficolin to elute at an ionic strength comparable with that of peak 2. Peak 2 was pooled an analyzed by size chromatography and SDS-PAGE. Fig. 4C shows size chromatography of the purified L-ficolin and of serum where the fractions were analyzed for L-ficolin. The size of the purified L-ficolin was found to be ϳ650 kDa and in accordance with the size observed for L-ficolin in serum. Serum also revealed a minor peak of L-ficolin eluting at 150 kDa, likely representing lower oligomeric forms.
Analysis of ion exchange peak 2 by SDS-PAGE and Western blotting is shown in Fig. 5A. Duplicate gels were run; one was silver-stained, whereas the other was blotted and developed with polyclonal rabbit anti-L-ficolin antibody. When the sample was analyzed under reducing conditions, a band of 35 kDa representing the polypeptide chain of L-ficolin appeared upon silver staining (Fig. 5A, lane 1). When the sample was analyzed under non-reducing conditions, four bands appeared, two Ͼ250 kDa, one ϳ35 kDa, and a weak band ϳ45 kDa (lane 2). The bands above 250 kDa and the band around 35 kDa were identified as L-ficolin when an identical gel was blotted and probed with anti-L-ficolin antibody. The high molecular mass bands represent L-ficolin oligomers, whereas the band at 35 kDa represents non-covalently associated L-ficolin peptide chains. The weak band around 45 kDa (lane 4) was not developed by anti-L-ficolin antibodies and was not identified.
L-ficolin obtained by the present purification method was compared with L-ficolin prepared by the previously published method (Fig. 5B) (14). In Fig. 5B the odd numbered lanes show L-ficolin prepared as previously described, and the even numbered lanes show the present preparation of L-ficolin. Lanes 1 and 2 were developed with anti-L-ficolin antibody. The two bands appearing in the present preparation correspond in size to the L-ficolin peptide chain (35 kDa) and the oligomeric complex (Ͼ250 kDa). In lane 1 bands appear at around 35, 200, and 250 kDa and one larger than 250 kDa. The 35-kDa band corresponds to the L-ficolin peptide chain, whereas the band above 250 kDa represents the fully covalently linked oligomeric L-ficolin. On a high resolution gel, as in Fig. 5A, this Ͼ250-kDa band may spilt up into two bands. The bands at 200 and 250 kDa may be degradations products of oligomeric L-ficolin. In our preparation (lane 2) only the largest band of more than 250 kDa and the 35-kDa band appear. The relative amounts of the lower order oligomers increased upon storage at 4°C, indicating proteolytic degradation, and no such degradation was observed upon storage of L-ficolin purified according to the present procedure. L-ficolin purified from serum by analytical affinity chromatography on antibody-coated microtiter wells and analyzed immediately afterward showed a distribution identical to that of L-ficolin purified by the present procedure (not shown). The band at 35-kDa seen in all preparations presumably represents non-covalently linked single L-ficolin polypeptide chains.
We have previously experienced that the MASPs dissociate from MBL in the presence of EDTA only at high ionic strength (25), and experiments on gel permeation chromatography on serum revealed that both EDTA and high salt concentration is required to release MASP-2 from complexes (27). To analyze directly the interaction with ficolin, we studied the influence of NaCl concentrations on L-ficolin-MASP-2 and H-ficolin-MASP-2 complexes. The ficolin-MASP complexes from serum were bound onto antibody-coated microtiter wells. The serum was diluted in buffers of various ionic strengths followed by the detection of MASP-2 with europium-labeled monoclonal anti-MASP-2 antibody. We found that MASP-2 was eluted from L-ficolin and H-ficolin in buffers containing 500 mM NaCl and EDTA but not when the buffers contained 500 mM NaCl and calcium. In accordance with this, the procedure described here involving the use of EDTA-containing high salt buffer yielded L-ficolin devoid of MASPs as determined by Western blotting (Fig. 5B). On the other hand L-ficolin prepared by the previously described method (14) contained MASPs. With anti-MASP-1/3 antibody (Fig. 5B, lane 3 and 4)

DISCUSSION
The specificity of the two pattern recognition molecules, Lficolin and H-ficolin, was examined and also compared with the results of our previous investigation of MBL. The results show striking differences in ligand selectivity. MBL is inhibited by all the sugars with horizontal C3-OH and C4-OH groups. As previously reported L-ficolin was inhibited by GlcNAc and Gal-NAc (18). However, the inhibition by the acetyl group was found to be independent of the hexose structure. Acetyl groups attached to a hexose ring (N-acetylmannosamine, GlcNAc, and GalNAc) as well as acetyl groups on cysteine, glycine, and choline were all efficient inhibitors (Table I). Ma et al. (28) recently described the binding of L-ficolin to ␤-1,3 glycans (curdlan). However, the precise ligand structure was not identified (28). Another unidentified L-ficolin binding structure is generated by CNBr activation of Sepharose beads (29).
The binding of H-ficolin was not inhibitable by any of the compounds examined. GlcNAc has previously been reported to inhibit the binding of H-ficolin to lipopolysaccharides (19), but the binding to A. viridans, which we studied, was unaffected by 100 mM GlcNAc. Binding of H-ficolin to A. viridans could only be inhibited by incubation with an A. viridans-derived polysaccharide (30).
The fbg domain of L-ficolin is expected to contain the ligand binding motif. The fbg domain is an ancient motif also found in the invertebrate taxa. In the horseshoe crab Tachypleus tridentatus a group of proteins named tachylectins (TLs) presents fbg domains (31). The various TLs are believed to be involved in innate immunity since they bind to pathogen-associated molecular patterns (32)(33)(34)(35)(36). TL-5A and TL-5B have molecular masses of ϳ41 kDa under reducing conditions, whereas in native state they are oligomers with an M r of 160 -300 kDa, i.e. broadly similar to human ficolins. The hemagglutinating activity of TL-5A and TL-5B was calcium-dependent, i.e. inhibitable by EDTA, and it was also inhibited by acetylated compounds like acetylsalicylic acid, acetylcholine, and acetyl coenzyme A (36). The primary structure of the fbg domain in human ficolins shows similarity to that of TL-5A (Fig. 6A) (20), and it is, thus, likely that one may gain useful information by using the crystal structure of TL-5A as a model for the fbg domain of the human ficolins (Fig. 6B). Two regions of TL-5A are functionally important; they are the calcium binding site and the ligand binding site. A calcium ion is bound by , and Thr-199 (Fig. 6B). Two water molecules are also involved. The calcium binding site is connected to the ligand binding site by a disulfide bridge between Cys-204 and Cys-217, which may , inhibition of binding to GlcNAc beads, were determined graphically from inhibition curves as shown in Fig. 1 except for MBL, which was taken from Haurum et al. (42). The relative inhibitor potential ("Relative") was determined by dividing the I 50 of the best inhibitor with the I 50 of the desired compound. NI, not inhibitory (i.e. resulting in less than 50% inhibition at 100 mM).  3. Binding of L-ficolin to CysNAc beads. L-ficolin was quantified in the supernatant after incubation of the 4 -8% PEG cut with beads at varying conditions, and the percentage L-ficolin bound to the beads was calculated. L-ficolin binding was studied in the presence of calcium (2 mM) or EDTA (5 mM) and at the varying NaCl concentrations given on the x axis. explain the calcium dependence of the ligand binding. The ligand binding site has a hydrophobic funnel composed of side chains from Tyr-208, His-218, Tyr-234, and Tyr-246, whereas the side chain of Ala-235 is situated at the base and closes the bottom of the funnel. TL-5A was crystallized in complex with GlcNAc (20). The acetyl group was found inside the funnel with van der Waals contact to Ala-235. Outside the funnel a cispeptide bond between Arg-216 and Cys-217 creates a sharp turn that enables hydrogen bond formation between the nitrogen in GlcNAc and the backbone His-218 and Tyr-234 of TL-5A. The hexose ring structure is recognized by Arg-184, which interacts with the C1-OH group, and Tyr-208 (coordinating a water molecule) interacts with the C3-OH group (20).
In the following comparison of the primary structure of TL-5A and the human ficolins we use the numbering from TL-5A (Fig. 6B). The calcium binding site amino acids Asp-194 and Asp-196 are conserved in all four proteins. The amino acid corresponding to His-197 is conserved in H-ficolin but substituted with an Asn in L-ficolin and M-ficolin. The last of the amino acids involved in coordinating the calcium ion in TL-5A, Thr-199, is not found in any of the human ficolins. In L-ficolin and M-ficolin it is substituted with leucine and valine, respectively, which are hydrophobic amino acids of similar size to threonine, whereas in H-ficolin it is substituted with serine, which is similar in nature to threonine but smaller. Because it is the backbone carbonyl oxygens that are involved in the calcium coordination, the substitutions of His-197 and Thr-199 might not affect the calcium coordinating ability. This suggests that L-ficolin, M-ficolin, and H-ficolin, like TL-5A, are calciumdependent. In agreement with this we found the binding of L-ficolin to CysNAc beads to be calcium-dependent at physiological ion strength. On the other hand under non-physiological high NaCl concentrations L-ficolin bound efficiently to the Cys-NAc beads in the presence of EDTA (Fig. 3). It, thus, seems possible that the substitutions in L-ficolin may disrupt the polarity of the binding site, making it calcium-independent at some conditions.
In the ligand binding site TL-5A, L-ficolin and M-ficolin have a funnel comprised of four aromatic side chains, two or one phenylalanine, one histidine, and one or two tyrosine residues, respectively, and an alanine at the base. The Arg-184 coordinating the C1-OH group of GlcNAc is absent in L-ficolin and M-ficolin, and Tyr-208, involved in coordination of the C3-OH group by interaction with a water molecule, has been substituted by phenylalanine, which is incapable of interacting with a water molecule. L-ficolin and M-ficolin appear to have lost the residues involved in the recognition of carbohydrates, suggesting that these two ficolins target acetylated compounds relatively independently of the structure of the acetylated molecule. This would agree with our results and indicates that L-ficolin should not be grouped as a lectin if the term lectin is used in the traditional way meaning that carbohydrates are the preferred ligands.
Recently the structure of a trimer of the fbg domain of recombinant human L-ficolin produced in insect cells in complex with GlcNAc was solved by x-ray crystallography and presented at the XXth International Complement Workshop (37). The structure revealed that the ligand binding site of L-ficolin is situated at a position different from that seen in tachylectin, approximately at the opposite side of the globular domain, thus also distant from the calcium binding site. This unexpected structure calls for caution when trying to deduce functionalities from orthologous structures. Through evolution a useful basic domain may be exploited for any number of functions as is well illustrated by the diversity of proteins of the immunoglobulin superfamily (38) by the existence of the so-called C-type lectin domain in proteins, which are devoid of lectin function (39), and by the fbg domain, serving quite distinct functions in fibrinogen and in tachylectin. L-ficolin may, thus, have diverged so much from tachylectin as to be no longer a lectin but an acetyl binding molecule, the biological functions of which remains to be revealed.
When comparing the potential ligand binding site H-ficolin differs significantly from TL-5A as well as from the other two human ficolins. It has undergone more substitutions in the hydrophobic funnel than L-ficolin and M-ficolin, and some of the hydrophobic amino acids have been substituted with polar and charged residues. This may explain why H-ficolin binding to A. viridans cannot be inhibited by any of the acetylated compounds examined. Further investigations are needed to determine the selectivity of H-ficolin because so far only one compound, a preparation of capsular structures from A. viridans (polysaccharides) (30), has been observed to inhibit Hficolin, but the ligand structure is unknown. One is anxiously awaiting the crystal structure of H-ficolin.
Using the knowledge gained from the inhibition studies, we developed an L-ficolin purification strategy substituting the previously used GlcNAc beads (14) with CysNAc-derivatized beads for affinity chromatography. By using CysNAc beads and by loading the sample in the presence of EDTA and 500 mM NaCl, we prevent binding of MBL to the beads and further disrupt the L-ficolin-MASP complexes. The elution with 20 mM NaCl allows for direct further processing on an ion exchange column. The present and the previous procedures (14) result in an almost identical composition concerning the oligomeric state of the purified L-ficolin with the difference that the product obtained by a previous procedure (14) contains some lower molecular weight L-ficolin, the proportion of which we have observed to increase upon storage.
We have previously found that the binding between MASPs and MBL can be disrupted by the presence of high NaCl concentrations in the presence of EDTA (25). In agreement with Cseh et al. (27) we found that the interaction between L-ficolin and MASP-2 is inhibited by high NaCl and EDTA. Because of the use of 500 mM NaCl and EDTA in the affinity chromatography procedure, the present procedure yields a MASP-free product, whereas the previous procedure yields L-ficolin-MASP complexes. When analyzed in its native state by size permeation chromatography, the purified L-ficolin elutes at a position identical to L-ficolin in serum. One might have expected an increased apparent size of L-ficolin in serum since it associates with MASPs. However, the elution profile of L-ficolin does not fit that of a globular protein but shows a higher Stokes radius, and there is no immediate reason why binding of MASPs should change this significantly. Similar analyses of MBL also failed to reveal a change in elution when complexed with MASPs (40).
Although in this discussion we have interpreted the effect of high sodium chloride concentration as the result of high ionic strength, it is certainly possible that the observation may reflect a more direct interaction of the sodium ion with the proteins (41). Further experiments should be aimed at elucidating this problem with respect to the binding of Lficolin to ligands as well as with respect to the formation of ficolin-MASP complexes.
The elucidation of the precise ligand structure for the pattern recognition molecules, L-ficolin and H-ficolin, is of obvious importance for illuminating their role in the immune defense as well as their possible participation in endogenous homeostatic mechanisms. The purification procedure described herein will allow for the production of L-ficolin for such studies.