Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen)1 Misao Matsushita,2* Mikio Kuraya,* Naotaka Hamasaki,† Mitsushi Tsujimura,‡ Hiroshi Shiraki,‡ and Teizo Fujita* Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBLassociated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway. The Journal of Immunology, 2002, 168: 3502–3506.
F
icolins are a group of proteins which consist of collagenlike and fibrinogen-like domains (1), and were originally identified as TGF--1-binding proteins on porcine uterus membranes (2, 3). They have been identified in both vertebrates and invertebrates so far (1). There is considerable evidence showing that ficolins are lectins which have in common a specificity for N-acetylglucosamine (GlcNAc)3 (4 –9). The fibrinogen-like domain of ficolins is likely to be responsible for exhibiting their lectin activity (10). In humans, two kinds of serum ficolins, Lficolin/P35 (4) and H-ficolin (5), and one kind of membrane-associated ficolin expressed in lung and blood cells have been identified and characterized (11, 12). The human serum ficolin L-ficolin/P35 is a multimeric protein consisting of 35-kDa subunits. In addition to being a GlcNAc-binding lectin (4), it also binds to elastin (13) and corticosteroid (14). The L-ficolin/P35 functions as an opsonin when it binds certain types of oligosaccharides on the surfaces of pathogens via its lectin activity (4). H-ficolin (also called Hakata Ag or thermolabile -2 macroglycoprotein) was first identified in serum as an Ag which is recognized by an autoantibody in patients with systemic lupus erythem-
*Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan; †Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and ‡ Fukuoka Red Cross Blood Center, Chikushino, Japan Received for publication December 21, 2001. Accepted for publication January 30, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Japan Society for the Promotion of Sciences (to M.M. and T.F.) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.M. and T.F). 2 Address correspondence and reprint requests to Dr. Misao Matsushita, Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan. E-mail address:
[email protected] 3 Abbreviations used in this paper: GlcNAc, N-acetylglucosamine; MBL, mannosebinding lectin; MASP, MBL-associated serine protease; sMAP, small MBL-associated protein; PSA, polysaccharide from Aerococcus viridans.
Copyright © 2002 by The American Association of Immunologists
atosus (15). H-ficolin forms oligomers consisting of 34-kDa subunits linked by disulfide bonds, like L-ficolin/P35, and shows a ladder of ⬎10 bands on SDS-PAGE under nonreducing conditions (16). The primary structure of H-ficolin has the characteristics of the ficolin family (5), consisting of an NH2-terminal region of 24 aa, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 aa, and a fibrinogen-like domain of 207 aa. Its mRNA is found in liver and lung (17). H-ficolin binds to GlcNAc and N-acetylgalactosamine; therefore, it agglutinates human erythrocytes coated with LPS derived from Salmonella typhimurium, Salmonella minnesota, and Escherichia coli (O111; Ref. 5). Like ficolins, mannose-binding lectin (MBL), a member of the collectin family (18), is a serum-collagenous lectin which recognizes certain carbohydrates such as mannose and GlcNAc (19). MBL has several oligomeric forms with a subunit which has a carbohydrate-recognition domain as well as a collagen-like domain. Human MBL is complexed through its collagen-like domain with three types of C1r/C1s-like serine proteases, termed MBLassociated serine proteases (MASP) (20), MASP-1 (21–23), MASP-2 (24), and recently described MASP-3 (25). MBL is also associated with small MBL-associated protein (sMAP; also called MAp19), which is a truncated form of MASP-2 (26, 27). The composition of MASPs and sMAP in MBL-MASP complexes varies from oligomer to oligomer (25). MBL-MASP circulates in blood, and upon binding to pathogens via MBL, MASPs convert from an inactive proenzyme form consisting of a single polypeptide to an activated form containing two polypeptides linked by a disulfide bond, which has proteolytic activities. MASP-1 has proteolytic activities against C3 and C2 (28), MASP-2 cleaves C4 and C2 (24, 28, 29), and MASP-3 has inhibitory activity against MASP-2 (25). The function of sMAP in the complex is not known. Complement activation by MBL-MASP is called the lectin pathway and is the third pathway of activation. In a previous paper, we demonstrated that L-ficolin/P35 is associated with MASP-1, MASP-2, and sMAP, and that L-ficolin/ P35-MASP complexes activate complement, as does MBL (30). The activation of complement by L-ficolin/P35-MASP implies that 0022-1767/02/$02.00
The Journal of Immunology L-ficolin/P35 is a collagenous lectin capable of activating the lectin pathway. Thus, L-ficolin/P35 may play a role in innate immunity by acting as an opsonin and activating the lectin pathway like MBL. In the present paper, we show that H-ficolin is another ficolin associated with MASPs and sMAP that activates the lectin pathway like MBL and L-ficolin/P35.
Materials and Methods Isolation of H-ficolin, L-ficolin/P35-MASP, and MBL-MASP The pastes of Cohn III fraction from human plasma were kindly provided by the Chemo-Sero-Therapeutic Research Institute, Kaketsuken (Kumamoto, Japan). They were dissolved in 50 mM Tris, 200 mM NaCl, and 20 mM CaCl2, pH 7.8 (starting buffer). After clotting, the filtrate was precipitated with 8% polyethylenglycol 4000. The precipitates were dissolved in starting buffer and then applied to a GlcNAc-agarose column (Sigma Aldrich, St. Louis, MO). MBL-MASP was eluted with starting buffer containing 0.3 M mannose. L-ficolin/P35-MASP was then eluted with starting buffer containing 0.15 M GlcNAc. MBL-MASP was purified further with monoclonal anti-MBL (3E7)-Sepharose (21) and L-ficolin/P35-MASP was purified further using Mono Q (30). The pass-through fractions from the GlcNAc-agarose column were used to purify H-ficolin. They were applied to monoclonal anti-H-ficolin (4H5)-Sepharose equilibrated with 50 mM Tris, 200 mM NaCl, and 10 mM CaCl2 (pH 7.8). H-ficolin was eluted with 0.1 M Gly-HCl buffer (pH 2.2), and then neutralized with 1 M Tris-HCl buffer, pH 8.2 (5). After dialysis against 50 mM Tris, 200 mM NaCl, and 10 mM CaCl2 (pH 7.8), the H-ficolin preparations were applied to Lentil lectin-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) and eluted with 0.2 M ␣-methylmannopyranoside. To remove contaminating IgM and IgG in the H-ficolin preparations, they were passed through antiIgM-Sepharose and protein A-Sepharose (Amersham Pharmacia Biotech) columns. The H-ficolin preparations were then passed through anti-MBL (3E7)-Sepharose and anti-L-ficolin/P35 (GN4)-Sepharose (31).
Preparation of PSA, a polysaccharide produced by Aerococcus viridans PSA, a polysaccharide produced by A. viridans, was prepared as described previously (32).
Immunoblotting After SDS-PAGE (12% gel) under reducing conditions, proteins were transferred from the gels to polyvinylidene difluoride membranes and the blots were probed with Abs against MASPs. Abs against synthetic peptides representing the 19 C-terminal amino acids of MASP-1 (24) and the 19 N-terminal amino acids of MASP-3 (25) were provided by Dr. J. Jensenius (Aarhus University, Aarhus, Denmark). Ab against a synthetic peptide representing the 20 N-terminal amino acids of MASP-2/sMAP (30) was provided by Dr. I. Terai (Health Sciences University of Hokkaido, Sapporo, Japan). Peroxidase-conjugated secondary Abs were used and developed with a Konica Immunostaining HRP kit (Konica, Tokyo, Japan).
3503 (GN5). After blocking, H-ficolin preparations, MBL-MASP, or L-ficolin/ P35-MASP diluted with TBST (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, and 0.1% Tween 20, pH 7.5) were incubated at 37°C for 60 min in the wells. After washing the wells with TBST and then with MGVB, C4 was incubated at 37°C for 60 min in the wells. C4b deposition on the wells was detected by adding HRP-conjugated polyclonal anti-C4 and developing with ABTS. To assay C4 activation by H-ficolin preparations upon binding of H-ficolin to PSA, ELISA plates were first coated with PSA. After blocking, H-ficolin preparations diluted with TBST were incubated at 37°C for 60 min in the wells. The procedure for detecting C4b deposition on the wells was the same as that described above for C4 activation by Ab-bound H-ficolin preparations.
Results Presence of MASPs and sMAP in H-ficolin preparations H-ficolin was isolated from Cohn Fraction III using affinity chromatography on monoclonal anti-H-ficolin-Sepharose and Lentil lectin-Sepharose. To examine the H-ficolin preparations for the presence of MASPs and sMAP, they were finally passed through anti-L-ficolin/P35 and anti-MBL columns to ensure the absence of L-ficolin/P35-MASP and MBL-MASP (Fig. 1). The L-ficolin/P35MASP and MBL-MASP were also prepared from Cohn Fraction III as described in Materials and Methods. As shown in Fig. 2, lane 1, Western blotting analysis revealed that MASP-1 (L chain), MASP-2 (H chain), MASP-3 (L chain), and sMAP were present in the H-ficolin preparations as in the preparations of L-ficolin/P35MASP and MBL-MASP. Because the H-ficolin preparations were made using mAb against H-ficolin, the presence of MASP-1, MASP-2, MASP-3, and sMAP in the H-ficolin preparations indicates that H-ficolin was complexed with these components (H-ficolin-MASP). Proteolytic activities of MASPs in H-ficolin-MASP We next determined whether MASPs in the H-ficolin-MASP complexes were proteolytically active. MASPs are known to have proteolytic activities against C4, C2, and C3. To examine C4 activation, H-ficolin-MASP was incubated with C4 and the residual C4 hemolytic activity was determined. H-ficolin-MASP consumed C4 in a dose-dependent manner (Fig. 3A). C4 activation is ascribed to the proteolytic activity of MASP-2, but C1s is another serine protease which can cleave C4. To exclude the possibility that C4 consumption was mediated by C1s and not by MASP-2, the effect of anti-C1s serum on C4 consumption was determined. It was
Assay of MASP activity The following buffers were used for the assays of MASP activity. MGVB is a low ionic strength veronal-buffered saline containing 0.1% gelatin, 2.3% mannitol, 2 mM CaCl2, and 0.5 mM MgCl2 (pH 7.5). EDTA-GVB is veronal-buffered saline supplemented with 10 mM EDTA and 0.1% gelatin. For the C4 consumption assay, human C4 was incubated at 37°C for 30 min with H-ficolin preparations. The residual C4 activity was then determined hemolytically. The average number of hemolytic sites per cell (z) was calculated as: z ⫽ ⫺ln (1 ⫺ y), where y is the hemolytic rate. The percentage of C4 consumed was calculated from z. To test the effect of anti-C1s on C4 consumption, H-ficolin preparations or human C1s were incubated at 37°C for 30 min with C4 in the presence of heat-inactivated polyclonal anti-C1s goat serum (Behringwerke, Marburg, Germany), and residual C4 activity was determined. For the C2 activation assay, Ab-sensitized sheep erythrocytes bearing guinea pig C1q and human C4b were incubated at 30°C for 15 min with H-ficolin preparations and oxidized human C2 in MGVB. Guinea pig serum diluted with EDTA-GVB was added to the reaction mixtures and they were incubated at 37°C for 60 min, after which EDTA-GVB was added, and z was determined. For the C3 cleavage assay, human C3 and H-ficolin preparations were incubated at 37°C for 60 min in VB-Ca. The reaction mixtures were then subjected to SDS-PAGE (7.5% gel) under reducing conditions. To assay C4 activation by the sold phase lectin-MASP complexes, ELISA plates were coated with monoclonal anti-H-ficolin (4H5), anti-MBL (3E7), or anti-L-ficolin/P35
FIGURE 1. SDS-PAGE profiles of H-ficolin preparations, L-ficolin/ P35-MASP, and MBL-MASP. H-ficolin, L-ficolin/P35-MASP, and MBLMASP were purified from Cohn Fraction III, as described in Materials and Methods. They were subjected to SDS-PAGE (12% gel) under reducing conditions and stained with Coomassie brilliant blue R-250. Molecular size markers are indicated on the left. Lane 1, H-ficolin preparations; lane 2, L-ficolin/P35-MASP; lane 3, MBL-MASP. Major bands in lanes 1, 2, and 3 represent the lectin component of each preparation. Upper, middle, and lower arrows indicate the H chain of MASP-1, the L chain of MASP-1, and sMAP, respectively. Bands for the other components (MASP-2 and MASP-3) are not detectable due to a low concentration.
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H-FICOLIN ACTIVATES THE LECTIN COMPLEMENT PATHWAY
FIGURE 2. Presence of MASPs and sMAP in H-ficolin preparations. H-ficolin (lane 1), L-ficolin/P35-MASP (lane 2), and MBL-MASP (lane 3) were subjected to SDS-PAGE under reducing conditions and then blotted. Blots were probed with Abs against MASP-1 (left), MASP-2/sMAP (middle), or MASP-3 (right). L and H represent the L chain of MASP-1 and the H chain of MASP-2, respectively. The lower bands of the L chain of MASP-1 and the H chain of MASP-2 represent cleaved products of MASP-1 and MASP-2, respectively.
found that it did not inhibit C4 consumption by H-ficolin-MASP under the conditions in which C4 consumption by C1s was completely inhibited (data not shown). This indicates that C1s is not involved in C4 activation by H-ficolin-MASP. C2 activation by H-ficolin-MASP was assessed by the formation of a C3 convertase, C4b2a, on C4b-bearing erythrocytes. As shown in Fig. 3B, C4b-bearing erythrocytes were hemolyzed by incubating them with H-ficolin-MASP and C2 followed by the addition of C3 to C9, indicating C2 activation by H-ficolin-MASP. The proteolytic activity of H-ficolin-MASP against C3 was assessed by SDSPAGE. H-ficolin-MASP cleaved C3 to generate the ␣⬘-chain of C3b in a dose-dependent manner (Fig. 3C). These results indicate that MASPs present in the H-ficolin-MASP complexes could cleave C4, C2, and C3 in the fluid phase. Complement activation by H-ficolin-MASP bound to Ab against H-ficolin We next determined whether H-ficolin-MASP could activate complement when the complex was bound to anti-H-ficolin Ab. H-ficolinMASP was incubated with anti-H-ficolin, anti-L-ficolin/P35, or antiMBL mAbs coated on ELISA plates. Similarly, L-ficolin/P35-MASP and MBL-MASP were incubated with the three kinds of mAbs as a
FIGURE 4. C4 activation by H-ficolin-MASP bound to anti-H-ficolin. H-ficolin-MASP, L-ficolin/P35-MASP, or MBL-MASP was reacted with anti-H-ficolin, anti-L-ficolin/P35, or anti-MBL mAb that had been coated on ELISA plates. After C4 was incubated in the wells, C4b deposition on the plates was evaluated. Data are mean ⫾ SD (n ⫽ 3).
control. C4 was then added to the wells, and after further incubation, C4b deposition on the plates was assessed. As shown in Fig. 4, Hficolin-MASP activated C4 when H-ficolin was bound only to antiH-ficolin. Similarly, L-ficolin/P35-MASP and MBL-MASP activated C4 when they were bound only to their corresponding Abs. Although H-ficolin-MASP shows ⬃3-fold increase in the amount of C4 deposited compared with the deposition of C4 in the presence of L-ficolin/ P35-MASP or MBL-MASP, these three types of lectin-MASP complexes are not comparable in efficiency in the activation of C4, since the amount of the lectin-MASP complexes bound to Ab might not be the same. The results shown in Fig. 4 confirm the formation of complexes between H-ficolin and MASP-2, and also show that the Hficolin-MASP preparations did not contain L-ficolin/P35-MASP or MBL-MASP. Complement activation by H-ficolin-MASP upon binding polysaccharide H-ficolin has been shown to bind to PSA, a polysaccharide produced by A. viridans (32). PSA consists of repeating units of glucose, mannose, GlcNAc, and xylose. Although the sugar residue(s) responsible for H-ficolin binding has not been identified, it is likely that H-ficolin binds to PSA via the lectin activity of its fibrinogenlike domain. We determined whether H-ficolin-MASP activates complement upon binding PSA. ELISA plates were first coated
FIGURE 3. Proteolytic activation of complement components by H-ficolin-MASP. A, C4 consumption. After incubation of C4 with the indicated amounts of H-ficolin-MASP, residual C4 hemolytic activity was determined and the percent inhibition caused by H-ficolin-MASP was calculated. B, C2 activation. C2 activation by the indicated amounts of H-ficolin-MASP was assayed as described in Materials and Methods. C, C3 cleavage. After incubation of C3 with various amounts of H-ficolin-MASP (lane 1, none; lane 2, 30 g/ml; lane 3, 15 g/ml; lane 4, 7.5 g/ml), the reaction mixtures were subjected to SDS-PAGE under reducing conditions.
The Journal of Immunology
FIGURE 5. C4 activation by H-ficolin-MASP bound to PSA. H-ficolinMASP, at the indicated amounts, was reacted with PSA that had been coated on ELISA plates. After C4 was incubated in the wells, C4b deposition on the plates was evaluated.
with PSA and then H-ficolin-MASP was incubated in the wells. C4 was then added to the wells, and after incubation, C4b deposition was assessed. As shown in Fig. 5, H-ficolin-MASP bound to PSAactivated C4. This indicates that upon binding to its ligand, Hficolin activates complement in association with MASP.
Discussion In a previous paper, we reported that like MBL, L-ficolin/P35 is associated with MASP-1, MASP-2, and sMAP, and that the complexes activate complement (30). As demonstrated in this paper, L-ficolin/P35 is also associated with MASP-3, which has been identified as a third serine protease present in the MBL-MASP complex (25). We showed here that, like L-ficolin/P35, H-ficolin is another lectin in the ficolin family that activates complement in association with MASP-1, MASP-2, MASP-3, and sMAP. Thus, it is concluded that three types of human serum lectin can activate complement in association with MASPs and sMAP; MBL of the collectin family, and L-ficolin/P35 and H-ficolin of the ficolin family. Complement activation by MBL-MASP was originally termed the lectin pathway, as the third pathway of activation of the complement system. However, based on recent findings, including those in this report, we propose that the concept of the lectin pathway should be expanded to include complement activation by a complex of a lectin and MASP. C1q, a subcomponent of C1 of the classical pathway, MBL, L-ficolin/P35, and H-ficolin are all oligomers and are similar in that they have a collagen-like domain within their subunits. These collagenous proteins are complexed with serine proteases which belong to the MASP/C1r/C1s family. Although it is most likely that the collagen-like domains of the four collagenous proteins are crucial for the binding of serine proteases (33), C1q differs from the other three collagenous proteins in that it is associated with C1r and C1s (34), and the others are associated with MASPs. The restricted binding specificities of the two types of serine proteases of the MASP/C1r/C1s family (C1r/C1s and MASPs) for the four collagenous proteins raises the possibility of a regulatory mechanism involved in complex formation. Although the lectin and the classical pathways in part share the activation mechanism, recent studies revealed that the MBL-mediated lectin pathway has unique features not possessed by the classical pathway. Three types of MASPs have been identified as serine proteases associated with MBL, while C1q is complexed with two types of serine proteases
3505 (C1r and C1s). MBL is also associated with a truncated form of MASP (sMAP), while the C1 complex does not contain such a truncated protein. MBL forms several types of oligomers and the composition of MASP binding to MBL varies from oligomer to oligomer (25). In contrast, C1q forms a hexameric oligomer and each oligomer is associated with C1r and C1s. The ficolins-mediated lectin pathway resembles the MBL-mediated lectin pathway in that like MBL, both L-ficolin/P35 and H-ficolin are complexed with three types of MASP and sMAP. Size heterogeneity was also found in human serum ficolins, especially in H-ficolin, suggesting a distinct MASP composition among the ficolin oligomers as is seen in the MBL oligomers. As we demonstrated in this paper, one of the functions of human serum H-ficolin would be to activate the complement system through the lectin pathway. H-ficolin binds to PSA produced by A. viridans and activates complement. Therefore, it is conceivable that like the other lectins capable of activating the lectin pathway, H-ficolin plays a role in host defense against pathogens. H-ficolin is produced not only in liver, but also in lung. The functions of H-ficolin produced in lung remain to be elucidated. However, we can speculate that H-ficolin in lung, as well as serum H-ficolin, might act as an opsonin, as is the case with L-ficolin/P35 present in human serum. If this is the case, H-ficolin plays an important role in innate immunity in both blood and lung. Both L-ficolin/P35 and H-ficolin bind to GlcNAc. Although studies comparing their precise specificities have not yet been conducted, their binding specificities appear to not be the same, because much smaller amounts of H-ficolin are obtained in the eluates with GlcNAc from the column compared with L-ficolin/P35 when purified using a GlcNAc column (data not shown). Therefore, the three collagenous lectins identified to date which can activate the lectin pathway might have both overlapping and distinct specificities for carbohydrates. The presence of these lectins might enable the host to eliminate pathogens on which various types of carbohydrates are present. In conclusion, H-ficolin is the second lectin of the ficolin family which can activate the complement pathway in association with MASPs and sMAP. Our findings that two types of serum ficolins (L-ficolin/P35 and H-ficolin) are lectins that can activate the lectin pathway suggest that ficolins are a group of lectins that function in host defense. In this context, it is of particular interest to note that there are ficolins with lectin activity in the body fluid of ascidians (9). This suggests that ficolins have a crucial role in innate immunity, not only in vertebrates but also in invertebrates.
Acknowledgments We thank Drs. J. C. Jensenius and I. Terai for providing anti-MASP Abs, and A. Nozawa for technical assistance.
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