Lectin-Associated Serine Protease Complex of Ficolin and Mannose

Cutting Edge: Complement-Activating Complex of Ficolin and Mannose-Binding Lectin-Associated Serine Protease

This information is current as Misao Matsushita, Yuichi Endo and Teizo Fujita of September 24, 2021. J Immunol 2000; 164:2281-2284; ; doi: 10.4049/jimmunol.164.5.2281 http://www.jimmunol.org/content/164/5/2281 Downloaded from

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Cutting Edge: Complement-Activating Complex of Ficolin and Mannose- Binding Lectin-Associated Serine Protease1

Misao Matsushita,2 Yuichi Endo, and Teizo Fujita

and characterized, and a third type of human ficolin has been Both ficolins and mannose-binding lectin (MBL) are lectins shown to be expressed in lung and blood cells (5, 6). The first characterized by the presence of collagen-like and carbohy-

serum ficolin has elastin-binding (7), corticosteroid-binding (8), Downloaded from drate-binding domains in a subunit, although their carbohy- and Escherichia coli-binding (9) activities, suggesting that it may drate-binding moieties are quite different. A fibrinogen-like be a multifunctional protein. We showed that this ficolin, termed domain is in ficolins, and a carbohydrate recognition domain is ficolin/P35 (previously called P35), is an N-acetylglucosamine in MBL. On binding to pathogens, human MBL activates the (GlcNAc)-binding lectin with opsonic activity (10). The collagen- complement system via the lectin pathway in association with like and fibrinogen-like domains of ficolin/P35 are likely to be two types of MBL-associated serine proteases (MASP), responsible for its opsonic and GlcNAc binding activities, respec- http://www.jimmunol.org/ MASP-1 and MASP-2 and its truncated form, small MBL- tively (11). The second ficolin from human serum, which is a lectin associated protein (sMAP, also called MAp19). We report here with binding specificity for GlcNAc/N-acetylgalactosamine/fu- that ficolin/P35, a human serum ficolin, was found to copurify cose, is termed Hakata Ag (12). with MASPs and sMAP. MASPs that were complexed with Serum MBL recognizes certain carbohydrates such as mannose ficolin/P35 exhibited proteolytic activities against complement and GlcNAc. MBL has a subunit which contains a collagen-like components C4, C2, and C3. The ficolin/P35-MASPs-sMAP domain and a carbohydrate recognition domain. In humans, C1s complex that was bound to Salmonella typhimurium activated MBL is complexed through its collagen-like domain with two complement. These findings indicate that ficolin/P35 is a sec- types of C1r/C1s-like serine protease, termed MBL-associated ond collagenous lectin capable of activating the lectin pathway serine protease (MASP) (13), MASP-1 (14–16) and MASP-2 (17). by guest on September 24, 2021 and thus plays a role in innate immunity. The Journal of Im- Like C1s MASP exhibits proteolytic activities against C4 and C2 munology, 2000, 164: 2281–2284. (14, 17), although the comparative efficiency of these proteases remains to be elucidated. Unlike C1s, MASP is able to cleave C3 (18). MBL is also associated with small MBL-associated protein nimal lectins prevent infection by pathogens through the (sMAP, also called MAp19) which is a truncated form of MASP-2 innate immune system in which they aggregate micro- (19, 20). The MBL-MASPs-sMAP complex circulates in blood organisms and in some cases act as an opsonin. Ficolins and upon binding to pathogens via MBL, MASPs convert from an A 3 (1) and mannose-binding lectin (MBL) (2) possess such func- inactive proenzyme form consisting of a single polypeptide to an tions. Ficolins are a group of proteins that consist of collagen-like activated form with two polypeptides linked by a disulfide bond, and fibrinogen-like domains and were originally identified as TGF- thus acquiring proteolytic activities. As a third activation pathway, ␤1-binding proteins on porcine uterus membranes (3, 4). To date, complement activation by MBL-MASPs-sMAP is termed the lec- ficolins have been identified in several kinds of vertebrates. In tin pathway (21). The structural and functional similarities be- humans, two kinds of ficolins present in serum have been isolated tween ficolin/P35 and MBL prompted us to investigate whether ficolin/P35 activates complement via the lectin pathway in a manner similar to that of MBL-MASPs-sMAP. Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan Received for publication November 8, 1999. Accepted for publication December 29 1999. Materials and Methods Isolation of ficolin/P35 and MBL-MASPs-sMAP 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 Human serum was precipitated with 7% polyethylene glycol 4000. The with 18 U.S.C. Section 1734 solely to indicate this fact. precipitates were dissolved in 50 mM Tris, 200 mM NaCl, 20 mM CaCl2 1 This work was supported by Grant-in-Aid for Scientific Research (C) 11670328 (pH 7.8) (starting buffer) and then applied to a GlcNAc-agarose column from the Ministry of Education, Science, Sports and Culture of Japan. (Sigma, St. Louis, MO). MBL-MASPs-sMAP was eluted with starting 2 Address correspondence and reprint requests to Dr. Misao Matsushita, Department buffer containing 0.3 M mannose. Ficolin/P35 was then eluted with starting of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikariga-oka, buffer containing 0.15 M GlcNAc. MBL-MASPs-sMAP was further puri- Fukushima 960-1295, Japan. E-mail address: [email protected] fied with monoclonal anti-MBL (3E7)-Sepharose (14). Ficolin/P35 was 3 Abbreviations used in this paper: MBL, mannose-binding lectin; GlcNAc, N-acetyl- further purified using Mono Q (Pharmacia, Piscataway, NJ) (10). Ficolin/ glucosamine; MASP, MBL-associated serine protease; sMAP, small MBL-associated P35 was finally passed through 3E7-Sepharose equilibrated with 50 mM protein. Tris, 200 mM NaCl, 10 mM CaCl2 (pH 7.8).

● 2282 CUTTING EDGE

Immunoblotting After SDS-PAGE (12% gel) under reducing conditions, proteins were transferred from gels to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and blots were probed with rabbit Abs against MASP-1 or MASP-2. Rabbit Abs against a synthetic peptide representing the19 C- terminal amino acids of MASP-1 (17), and the 20 N-terminal amino acids of MASP-2/sMAP were provided by Dr. J. Jensenius (Aarhus University, Aarhus, Denmark) and Dr. I. Terai (Hokkaido Institute of Public Health, Sapporo, Japan), respectively. Peroxidase-conjugated anti-rabbit IgG was used as a second Ab and was developed with a Konica Immunostaining HRP kit. (Konica, Tokyo, Japan). Immunoprecipitation Ficolin/P35 preparations were incubated with anti-ficolin/P35 (GN5) (22) or 3E7 in Veronal buffer containing 0.148 M NaCl and 10 mM CaCl2 (pH 7.4) at 4°C for 30 min and then with protein G-Sepharose for 30 min. Bound proteins were subjected to SDS-PAGE (12% gel) under reducing conditions and immunoblotted. To examine for the presence of a ficolin/ P35-MASPs-sMAP complex in human serum, human serum was incubated with GN5-Sepharose or Sepharose in the presence of 0.3 M mannose and 0.15 M GlcNAc at 4°C for 30 min. Mannose and GlcNAc were used to eliminate the possible binding of MBL and ficolin/P35 to the gels through Downloaded from a lectin activity. After incubation, bound proteins were subjected to SDS- PAGE and immunoblotted. Assay of MASP activity For the assays in this study, the following buffers were used. 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 http://www.jimmunol.org/ Veronal-buffered saline supplemented with 10 mM EDTA and 0.1% gelatin. C4 consumption was assayed as described (14). In brief, human C4 was incubated at 37°C for 30 min with ficolin/P35 preparations. The residual C4 activity was then determined hemolytically. The average number of hemolytic sites per cell (z) was calculated as z ϭϪln (1 Ϫ y) where y is hemolytic rate. The percentage of C4 consumption was calculated from z. To test the effect of anti-C1s on C4 consumption, ficolin/P35 preparations or human C1s (14) were incubated with C4 in the presence of heat- inactivated polyclonal anti-C1s rabbit serum (Behringwerke, Marburg, Germany) at 37°C for 30 min, and residual C4 activity was determined. For the C2 activation assay, Ab-sensitized sheep erythrocytes bearing guinea by guest on September 24, 2021 pig C1q and human C4b (EAC4b) were prepared as described (23). EAC4b cells, ficolin/P35 preparations and oxidized human C2 in MGVB were incubated at 30°C for 15 min. Guinea pig serum diluted with EDTA-GVB was added to the reaction mixtures and they were incubated at 37°C for 60 min. After incubation, EDTA-GVB was added and z was determined. For the C3 cleavage assay, human C3 and ficolin/P35 preparations were incubated in Veronal buffer containing 0.148 M NaCl and 10 mM CaCl2 (pH 7.4) at 37°C for 60 min. The reaction mixtures were then subjected to SDS-PAGE (7.5% gel) under reducing conditions. To assay C4 activation by ficolin/P35 bound to anti-ficolin/P35, ELISA plates were coated with monoclonal anti-ficolin/P35 (GN4 (22), GN5) or anti-MBL (3E7). After FIGURE 1. Association of ficolin/P35 with MASP-1, MASP-2, and blocking, ficolin/P35-MASP-sMAP diluted with TBST (50 mM Tris, 150 sMAP. A, Presence of MASP-1, MASP-2, and sMAP in ficolin/P35 prep- mM NaCl, 10 mM CaCl2, 0.1% Tween 20 (pH 7.5)) was incubated in the arations. Ficolin/P35 preparations (lane 1) or MBL-MASPs-sMAP (lane 2) wells at 37°C for 60 min. After the wells were washed with TBST and then were subjected to SDS-PAGE under reducing conditions followed by blot- with MGVB, C4 was incubated in the wells at 37°C for 60 min. C4 dep- ting. Blots were probed with Abs against MASP-1 (middle) or MASP-2/ osition on the wells was detected by adding biotinylated polyclonal anti-C4 sMAP (right). Blots were also protein stained (left). L and H represent the and peroxidase-conjugated avidin and developing with ABTS. To assay C4 activation by ficolin/P35 bound to Salmonella typhimurium, ficolin/P35- light chain of MASP-1 and the heavy chain of MASP-2, respectively. The MASPs-sMAP or MGVB was incubated with S. typhimurium TV119 at upper bands, which are reactive with anti-MASP-1 and with anti-MASP-2 4°C for 60 min. After the bacteria were washed with MGVB, they were in lane 2, are proenzyme MASP-1 and proenzyme MASP-2, respectively. incubated with human C4 at 37°C for 30 min. After the bacteria were B, Association of ficolin/P35 with MASP-1, MASP-2, and sMAP in fico- reacted with anti-C4 and FITC-conjugated anti-rabbit IgG, C4 deposition lin/P35 preparations. Ficolin/P35 preparations were incubated with anti- was analyzed with a FACScan (Becton Dickinson, Mountain View, CA). ficolin/P35 (GN5, lane 1) or with anti-MBL (3E7, lane 2) and then with protein G-Sepharose. Bound proteins were analyzed as in A. C, Association Results and Discussion of ficolin/P35 with MASP-1, MASP-2, and sMAP in serum. Human serum We purified ficolin/P35 from human serum using a GlcNAc col- was incubated with GN5-Sepharose (lane 3) or Sepharose (lane 4). The umn and Mono Q and finally an anti-MBL column to ensure the immunoprecipitates were subjected to immunoblotting analysis as in A. Lane 1, ficolin/P35 preparations; lane 2, MBL-MASPs-sMAP prepared in absence of MBL-MASPs-sMAP and looked for the presence of the proenzyme form of MASP. MASP-1, MASP-2, and sMAP in ficolin/P35 preparations by immunoblotting analysis. MASP-1 and MASP-2 in MBL-MASPs- sMAP isolated from human serum as a control were in their activated forms as shown by the stained bands of the L chain of chain), and sMAP were detected in ficolin/P35 preparations as in MASP-1 and the H chain of MASP-2 (Fig. 1A, lane 2). As can MBL-MASPs-sMAP, indicating that they contained the activated been seen in Fig. 1A (lane 1), MASP-1 (L chain), MASP-2 (H forms of MASP-1, MASP-2, and sMAP. The Journal of Immunology 2283

FIGURE 3. Complement activation by immobilized ficolin/P35- MASPs-sMAP. A, C4 activation by ficolin/P35-MASPs-sMAP bound to anti-ficolin/P35. Ficolin/P35-MASPs-sMAP was first reacted with anti-

ficolin/P35 (GN4 or GN5) or anti-MBL (3E7) on ELISA plates and C4 Downloaded from deposition on the plates was evaluated. B, C4 activation by ficolin/P35- FIGURE 2. Proteolytic activation of complement components by fico- MASPs-sMAP bound to S. typhimurium TV119. Bacteria were incubated lin/P35-MASPs-sMAP. A, C4 consumption. After incubation of C4 and with ficolin/P35-MASPs-sMAP (⅐⅐⅐⅐⅐) or buffer (OO) and then with C4. C4 various amounts of ficolin/P35-MASPs-sMAP, residual C4 hemolytic ac- deposition on S. typhimurium TV119 was analyzed by flow cytometry. tivity was determined and the percent inhibition caused by ficolin/P35- Fluoresence intensity (FL1-H) is plotted against the cell number. MASPs-sMAP was calculated. B, C2 activation. C2 activation by various amounts of ficolin/P35-MASPs-sMAP was assayed as in Materials and http://www.jimmunol.org/ Methods. C, C3 cleavage. After incubation of C3 with various amounts of ficolin/P35-MASPs-sMAP (lane 1, none; lane 2,50␮g/ml; lane 3,25 ficolin/P35-MASPs-sMAP complexes are the same as those of ␮g/ml; lane 4,12␮g/ml; lane 5,6␮g/ml), the reaction mixtures were MBL-MASPs-sMAP in a fluid phase. subjected to SDS-PAGE under reducing conditions. We next determined whether MASP in a solid phase ficolin/ P35-MASPs-sMAP complex activates complement. Ficolin/P35- MASPs-sMAP captured by anti-ficolin/P35 Ab coated on ELISA To determine whether MASP-1, MASP-2, and sMAP were plates was incubated with C4 and C4 deposition was assessed. As complexed with ficolin/P35, we immunoprecipitated ficolin/P35 shown in Fig. 3A, the solid phase ficolin/P35-MASPs-sMAP acti- preparations with anti-ficolin/P35 and analyzed the precipitates by vated C4. The results also indicate that MBL-MASPs-sMAP was by guest on September 24, 2021 immunoblotting. MASP-1, MASP-2, and sMAP coprecipitated not involved in this activation, because anti-MBL coated on the with ficolin/P35 when ficolin/P35 preparations were immunopre- plates did not cause C4 deposition. Ficolin/P35 binds S. typhi- cipitated with monoclonal anti-ficolin/P35 but not with anti-MBL murium TV119, and this binding is abolished by GlcNAc (10). To (Fig. 1B), indicating that MASP-1, MASP-2, and sMAP form a examine complement activation by ficolin/P35-MASPs-sMAP complex with ficolin/P35. To demonstrate directly the presence of bound to S. typhimurium TV119, S. typhimurium TV119 was first the ficolin/P35-MASPs-sMAP complex in human serum, human incubated with ficolin/P35-MASPs-sMAP and then with C4. C4 serum was adsorbed to anti-ficolin/P35-Sepharose, and the bound deposition on S. typhimurium TV119 was evaluated by flow cy- proteins were analyzed by immunoblotting. Proenzyme MASP-1, tometry. As shown in Fig. 3B, C4 deposition was noted with S. proenzyme MASP-2, and sMAP were recovered in the bound pro- typhimurium TV119 bearing ficolin/P35-MASPs-sMAP. teins (Fig. 1C). In this work, we demonstrated that ficolin/P35 is associated with Like C1, MASP-1 and MASP-2 which are associated with MBL MASP-1, MASP-2, and sMAP as is MBL. In blood, MASP-1 and activate C4 and C2. In addition, MASP-1 has a unique proteolytic MASP-2 might exist as inactive proenzyme forms complexed with activity against C3. To examine these proteolytic activities of ficolin/P35. MASP-1 and MASP-2 in ficolin/P35-MASPs-sMAP MASP in ficolin/P35-MASPs-sMAP, ficolin/P35-MASPs-sMAP complexes purified and analyzed in this study were in their acti- was incubated with C4, and the residual hemolytic activity of C4 vated forms. They may have been activated during purification, as was determined. As shown in Fig. 2A, ficolin/P35-MASPs-sMAP is seen with MBL-MASPs-sMAP. consumed C4 in a dose-dependent manner. This C4 consumption C1q, a subcomponent of C1 of the classical pathway, has a was not inhibited by anti-C1s serum under the conditions in which collagen-like domain with which C1r and C1s are associated. The C4 consumption mediated by C1s was completely inhibited, indi- presence of a collagen-like domain in MBL and the structural sim- cating that C1s was not involved in C4 consumption mediated by ilarities among MASP, C1r, and C1s enable MBL to bind C1r and ficolin/P35-MASPs-sMAP (data not shown). C2 activation by fi- C1s in vitro (24, 25). The proteolytic activity of ficolin/P35 prep- colin/P35-MASPs-sMAP was assessed by the formation of a C3 arations against C4 was not inhibited by anti-C1s, indicating that convertase, C4b2a, on C4b-bearing erythrocytes. The ficolin/P35- ficolin/P35 is not associated with C1s. Therefore, like MBL (26) MASPs-sMAP complex activated C2, resulting in the formation of the collagen-like domain of ficolin/P35 might also be crucial for C4b2a (Fig. 2B). The proteolytic activity of ficolin/P35-MASPs- the binding of MASPs and sMAP. sMAP against C3 was assessed by SDS-PAGE. After incubation of To date, many proteins with a fibrinogen-like domain, including ficolin/P35-MASPs-sMAP with C3, the mixtures were subjected tenascins (27) and the scabrous protein of Drosophila melano- to SDS-PAGE. Ficolin/P35-MASPs-sMAP cleaved C3 to generate gaster (28), have been reported, although the functions of the fi- the ␣Ј-chain of C3b in a dose-dependent manner (Fig. 2C). These brinogen-like domains have not been fully elucidated. Accumulat- results indicate that the proteolytic activities of MASP present in ing data, however, indicate that certain fibrinogen-like domains are 2284 CUTTING EDGE involved in recognition of microorganisms through a lectin activity 11. Le, Y., S. H. Lee, O. L. Kon, and J. Lu. 1998. Human L-ficolin: plasma levels, such as in ficolin/P35 and in the horseshoe crab lectins (29). 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