Pathway Activation of the Lectin Complement Serine Protease (MASP)

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Mannose-Binding Lectin (MBL)-Associated Serine Protease (MASP)-1 Contributes to Activation of the Lectin Complement Pathway This information is current as of September 28, 2021. Minoru Takahashi, Daisuke Iwaki, Kazuko Kanno, Yumi Ishida, Jie Xiong, Misao Matsushita, Yuichi Endo, Shigeto Miura, Naoto Ishii, Kazuo Sugamura and Teizo Fujita J Immunol 2008; 180:6132-6138; ; doi: 10.4049/jimmunol.180.9.6132 Downloaded from http://www.jimmunol.org/content/180/9/6132

References This article cites 24 articles, 12 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/180/9/6132.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mannose-Binding Lectin (MBL)-Associated Serine Protease (MASP)-1 Contributes to Activation of the Lectin Complement Pathway1

Minoru Takahashi,* Daisuke Iwaki,* Kazuko Kanno,* Yumi Ishida,* Jie Xiong,§ Misao Matsushita,† Yuichi Endo,* Shigeto Miura,‡ Naoto Ishii,§ Kazuo Sugamura,‡ and Teizo Fujita2*

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model Downloaded from .MASP1/3؊/؊) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway) Serum derived from MASP1/3؊/؊ mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2. The Journal of Immunology, 2008, 180: 6132–6138. http://www.jimmunol.org/

n the complement system, three pathways (classical, alterna- In the lectin pathway, mannose-binding lectin (MBL)3 or fico- tive, and lectin complement pathways) have been identified, lins recognize carbohydrates (such as mannose and N-acetylglu- I all of which are able to activate C3, the key component of cosamine) on microorganisms (5, 6). Activation of the pathway is complement (1–3). Activation of C3 is a crucial step in the com- triggered by serine proteases that are associated with MBL or fi- plement reaction, which further triggers the inflammation response colins, termed MASPs (MBL-associated serine proteases) (7). (for example, opsonization, anaphylatoxic shock, and membrane MASPs are similar to C1r and C1s in the classical pathway, and attack complex) to activate the later components from C5 to C9. In have highly conserved domain structures, including C1r/C1s/Uegf/ by guest on September 28, 2021 the classical pathway, the activation mechanism has been well bone morphogenetic protein 1, epidermal growth factor-like, C1r/ characterized: autoactivated C1r triggered by C1q that binds im- C1s/Uegf/bone morphogenetic protein 2, short consensus repeat 1, muno-complex activates C1s. Activated C1s then cleaves C4 and short consensus repeat 2, and serine protease domains. Three kinds C2 to form C3 convertase, C4b2a. In the alternative pathway, of MASP, MASP-1 (7), MASP-2 (8), MASP-3 (9), and a truncated

C3(H2O), which is spontaneously generated from circulating C3 in form of MASP-2, sMAP (10, 11), are present in MBL and ficolin the blood by contribution of water or ammonia at slow rate, is able complexes. Some in vitro evidence has suggested that MASP-2 to form a C3 convertase, C3(H2O)Bb, in the presence of factor D alone is sufficient to trigger complement activation. First, only and factor B. The alternative pathway has been understood to be an MASP-2 has proteolytic activity toward C4 and C2, similar to C1s amplification loop leading to the presence of much more C3b on in the classical pathway, although MASP-1 is able to cleave C2 (8, the surface of microorganisms with activated classical or lectin 12). Second, recombinant MASP-2 is easily activated by itself dur- pathways (4). ing purification (13), indicating that autoactivation of MASP-2 might occur in the lectin pathway cascade. However, the role of MASP-1 in the lectin pathway is still controversial; MASP-1 is capable of cleaving C3 directly (9, 14, 15), resulting in activation *Department of Immunology, Fukushima Medical University School of Medicine, of the alternative pathway (14). These results suggest that there Fukushima, †Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka, and ‡Department of Microbiology and Immunology, might be another route, independent of MASP-2, for the activation Tohoku University Graduate School of Medicine, Sendai, Japan; and §Department of of the complement system (14). However, it has not been con- Immunology and Laboratory of Allergy and Clinical Immunology, Institute of Al- firmed whether cleavage of C3 by MASP-1 would actually involve lergy and Immune-related Diseases and Medical Research Center, Wuhan University School of Medicine, Wuhan, China activation of the lectin pathway in vivo, as MASP-1 activity is very Received for publication July 24, 2007. Accepted for publication March 3, 2008. low (16). The function of MASP-3 is not yet clear. MASP3 mRNA 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. 3 1 Abbreviations used in this paper: MBL, mannose-binding lectin; MASP, MBL- This work was supported by Grant-in-Aids (11770078, 13770072, and 17790327) associated serine protease; ES, embryonic stem; sMAP, small MBL-assocaited pro- for Scientific Research from the Ministry of Education, Culture, Sports, Science, and tein; rMASP-1n, recombinant native type of MASP-1; rMASP-1i, recombinant inac- Technology of Japan, and by Core Research for Evolutional, Science, and Technol- tive MASP-1 mutant; rMASP-1K, recombinant MASP-1 mutant modified by ogy, Japan Science and Technology Agency. replacing the arginine429 with lysine; rMASP-2i, recombinant inactive MASP-2 2 Address correspondence and reprint requests to Dr. Teizo Fujita, Department of mutant. Immunology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 6133

were used according to the guidelines for animal experimentation of Fuku- shima Medical University.

RT-PCR for murine MASP3 and Gapdh Murine liver mRNA was purified by using PolyATract system 1000 (Pro- mega). First strand cDNA was synthesized from 1 ␮g of messenger RNA using the Ready-To-Go T-primed first strand kit (GE Healthcare Bio-Sci- ences). PCR was conducted using 1 ␮l of the first strand cDNA as template and the primer sets: 1F (5Ј-CAGAAGGAACCAAAGCCAGGAG-3Ј), 1R (5Ј-GCCCCACTCCCGAACCACTTGT-3Ј), and 2R (5Ј-GCGACCTC CGATGATTCTCTTC-3Ј) for MASP3, and 5Ј-GTATGTCGTGGAGTC TACTG-3Ј and 5Ј-TACTCCTTGGAGGCCATGTA-3Ј for Gapdh.

Recombinant murine MASP-1 To assess the enzymatic effect of MASP-1, two kinds of recombinant murine MASP-1 (recombinant native type of MASP-1 (rMASP-1n) and recombinant MASP-1 mutant modified by replacing the arginine429 with lysine (rMASP-1K)) have been expressed and purified in insect cells. Native FIGURE 1. Targeted disruption of the MASP1/3 gene. A, Schematic type of recombinant MASP-1 was defined as rMASP-1n. Mutant type of 429 representation of restriction maps of the wild-type MASP1/3 gene (upper), recombinant MASP-1 was defined as rMASP-1K in which the arginine Downloaded from the targeting vector (middle), and the mutated allele (lower). In the map was replaced with lysine. In addition, we used recombinant inactive 627 of the wild-type gene, the region from the first exon to the third is depicted; MASP-1 mutant (rMASP-1i) in which the serine was replaced with alanine. the closed boxes show exons. The translation initiation site is located in the Two recombinants, rMASP-1n and rMASP-1i were generated as fol- first exon. neo and DT-A indicate a neomycin-resistant gene and a diph- lows. The cDNA encoding the entire coding region of the MASP1 gene was theria toxin cassette, respectively. The restrictions sites are as follows: Xb, amplified by RT-PCR. A sequence encoding 6ϫ His was added at the XbaI; B, BamHI; Sl, SalI; and Sc, SacI. B, Southern blot analysis of prog- 3Ј-end of the cDNA instead of the native stop codon. The cDNA was ϩ Ϫ enies from MASP1/3 / intercrosses. Genomic DNA was digested with ligated into pVL1393 vector (BD Biosciences), to generate rMASP-1n. To http://www.jimmunol.org/ BamHI and hybridized with a BamHI-SalI fragment of genomic DNA, construct rMASP-1i, the codon for the serine residue at amino acid position including part of the first intron. The location of the probe is indicated with 627, essential for the active center of MASP-1, was replaced with an ala- bars under the wild and targeted alleles. nine using site-directed mutagenesis based on PCR. Both plasmid con- structs were transfected into Sf21 cells with BacPAK6 viral DNA (Bsu36I digest) using Bacfectin reagent (BD Biosciences). After incubation for 3 days at 27°C, recombinant virus was harvested from culture supernatants. is transcribed by an alternative splicing/polyadenylation mecha- Virus plaques were made by infection of virus into an agarose-fixed mono- nism from the MASP1/3 gene, in which the regions encoding the layer of Sf21 cells. Four plaques were picked up from each construct. serine protease domains of MASP-3 and MASP-1 are tandemly These virus stocks were further amplified by infection into Sf21 cells. For

located (9). expression of recombinant proteins, Sf21 cells were routinely cultured in by guest on September 28, 2021 500-ml Erlenmeyer flasks containing 200 ml of Sf900II serum-free me- In mouse, the MBL-MASP complex was first described as a dium (Invitrogen). For large scale preparation, 300 ml of Sf21 cells Ra-reactive factor that binds specifically to Ra chemotype strains (0.5ϫ106/ml) were infected with recombinant baculovirus by 5–10 multi- of Salmonella (17). Murine MASP-1 (18) and MASP-2 (19) have plicity of infection. To purify recombinant proteins, culture supernatant already been cloned and characterized. We previously described was concentrated by an Amicon stirred cell with YM-30 filter (Millipore) until Ͻ60 ml. Concentrated samples were dialyzed with HisTrap binding murine MASP3 cDNA from the livers of C57BL/6J mice (20). In buffer (20 mM TrisCl (pH 7.4), 0.5 M NaCl, 5 mM CaCl2, and 10 mM the present study, we generated MASP-1 and MASP-3 double- imidazole) and recombinant proteins were captured on HisTrap columns deficient mice using a standard gene targeting method. This knock- (GE Healthcare Bio-Sciences) at 0.5 ml/min. Captured proteins were eluted out mouse showed insufficient abilities to activate C3, C4, and by a gradient (from 0 to 30%) of elution buffer (containing 20 mM TrisCl (pH 7.4), 0.5 M NaCl, 5 mM CaCl , and 0.5 M imidazole). Fractions MASP-2. Furthermore these deficiencies have been restored by the 2 including MASP-1 were estimated by Western blotting using anti-MASP-1 recombinant MASP-1, suggesting that MASP-1 plays a crucial Ab and pooled fractions were dialyzed with TBS. role in direct or indirect activation of the lectin pathway. The procedure to make another recombinant, rMASP-1K, was described as below. Murine MASP1 cDNA that includes its full coding region and an Materials and Methods artificial histidine-tag at C terminus was subcloned in an expression vector, pIB/V5-His (Invitrogen). A codon that encodes the arginine residue at re- Generation of MASP1/3-deficient mice active site P1 for cleavage of MASP-1 between H and L chains was re- Genomic DNAs, including the murine MASP1/3 gene, were isolated from placed with a codon for lysine residue using a QuikChange kit (Stratagene). a 129Sv phage genomic library (Stratagene). The targeting vector was con- This vector was transfected in an insect cell line, High-Five by FuGENE structed as illustrated in Fig. 1A and transfected into J1 cells by electro- HD transfection reagent (Roche Diagnostic Systems). Stable transfectants poration. After selection with 200 ␮g/ml G418 (Invitrogen), embryonic were selected in Ex-Cell 405 medium (SAFC Biosciences) containing 10 ␮ stem (ES) clones were picked up, and their genotypes were analyzed by g/ml blasticidin (Invitrogen). Transfectants were further cultured in a Southern blotting using a BamHI-SalI fragment of genomic DNA as a suspension using an Erlenmeyer flask with shaking. To get recombinant Ͼ ϫ 6 probe. The expected homologous recombination was confirmed in four ES MASP-1K, 1.6 10 cells/ml transfectants was seeded in 200 ml of fresh ␮ ␮ clones. Two independent chimeric mice were generated by microinjection Ex-Cell 405 medium containing 1 g/ml aprotinin and 10 g/ml blastici- of two ES clones into C57BL/6J blastocysts. Germline transmissions were din. Approximately 1 milligram of rMASP-1K could be purified from 200 achieved in the offspring of both chimeric mice, by backcrossing with a ml of culture supernatant by binding to 1 ml of TALON resin (Clontech C57BL/6J line. The targeted locus was maintained by breeding of het- Laboratories). Protein concentration was estimated by BCA protein assay erozygous mice with wild-type C57BL/6J. Homozygous mice were gen- kit (Pierce). erated by mating of heterozygous pairs. The genotypes of mice were determined by PCR using tail genomic DNA as a template. Wild type-specific Expression and purification of recombinant murine MASP-2i alleles were amplified as 539 bp fragments using the primer set M1U: 5Ј-ACCCCTCCCTGCCTCAGACTGTTTGATA-3Ј and M1L: 5Ј-GCT Recombinant murine MASP-2i, in which the serine residue at the active GATGCTGATGTTAGGATGGTATTCT-3Ј; the mutant allele was de- center of the protease domain was replaced with an alanine, was expressed tected as 639 bp fragment by using M1L with another primer, NeoU: 5Ј- in Drosophila Schneider 2 cells and purified by a nickel column as de- CATCGCCTTCTATCGCCTTCTTGACGAGTT-3Ј. Mice in this study scribed (21). 6134 MASP-1 CAN ACTIVATE THE LECTIN PATHWAY

Polyclonal Abs against murine MASP-1 and MASP-2 Two kinds of polypeptide corresponding to the regional sequences of the L chain of MASP-1 (Glu584 to Asn680) and the H chain of MASP-2, including the sMAP region (Thr76 to Leu164), were expressed as recombinant proteins using a pET21a vector (Novagen) in Escherichia coli. The products were puriﬁed from a gel after SDS-PAGE by electro-elution (Bio-Rad). Polyclonal Abs were prepared by immunizing rabbits with the puriﬁed polypeptides as Ags. These Abs react with the denatured protein in SDS- PAGE. To establish a polyclonal Ab that recognizes native MASP-1, a rabbit was immunized with rMASP-1i.

Detection of MASP-1 in murine sera on mannan-coated plates Maxi-Sorp plates (Nunc) were coated with 100 ␮lof10␮g/ml mannan (M3640; Sigma-Aldrich) in sodium carbonate buffer (pH 9.6) soaked over-

night. Blocking was done by adding 1% BSA in TBS/5 mM CaCl2 (TBS/ Ca) buffer. After washing with TBS/Ca containing 0.05% Tween 20 (TBS/ Ca/tw), serially diluted murine serum in TBS/Ca/tw was added to the wells. After incubation for 30 min at 37°C, the wells were washed four times with TBS/Ca/tw. Each well was incubated with polyclonal anti-mouse MASP-1 for 30 min at room temperature, and then washed four times with TBS/ Ca/tw. The wells were further incubated with HRP-conjugated anti-rabbit Downloaded from Ig for 30 min. Enzymatic color development was achieved by 3,3Ј,5,5Ј- tetrametylbenzidine-Microwell Peroxidase Substrate system (KPL) and the

reaction was stopped by adding 1M H3PO4. Color was detected by the absorbance at 450 nm.

Western blotting of MBL-MASP complexes eluted from mannan-agarose http://www.jimmunol.org/ Mouse serum was diluted with TBS/Ca buffer. The diluted serum was incubated with 20 ␮l of mannan-agarose (Sigma-Aldrich). After washing the agarose four times with TBS/Ca/tw, the proteins captured on the agarose were eluted with 50 ␮lof1ϫ SDS-PAGE loading buffer with or without 2-ME. Eluted samples were separated by SDS-PAGE followed by FIGURE 2. MASP-1 and MASP-3 are completely absent in MASP1/ blotting to polyvinylidene fluoride membrane (Immobilon P; Millipore) in Ϫ/Ϫ ϩ/ϩ F ϩ/Ϫ Œ a semidry system (Bio-Rad). The blotted membranes were then subjected 3 serum. A, Mouse sera from MASP1/3 ( ), MASP1/3 ( ), and Ϫ/Ϫ to standard Western blotting. Briefly, the membrane was incubated with MASP1/3 (E) were incubated in mannan-coated microtiter wells at anti-MASP-1 or anti-MASP-2 Abs and then with HRP-conjugated anti- 37°C for 30 min. MASP-1 captured on wells was detected by rabbit anti- rabbit IgG (DakoCytomation). Proteins were detected using the ECL Plus MASP-1 and HRP-conjugated anti-IgG. B, A total of 20 ␮l of sera from by guest on September 28, 2021 Western blotting detection system (GE Healthcare Biosciences) and visu- MASP1/3ϩ/ϩ (lane 1), MASP1/3ϩ/Ϫ (lane 2), and MASP1/3Ϫ/Ϫ (lane 3) alized in a LAS3000 (Fuji Film). mice was incubated with mannan agarose for 30 min, and the bound fractions were subjected to Western blotting with anti-MASP-1 Ab under non- C4-deposition on mannan-coated microtiter plates reducing conditions. (p) and (a) indicate the zymogen and the active-form MaxiSorp surface of FluoloNunc plates (Nunc) were coated with 100 ␮lof of MASP-1, respectively. In lane 4, rMASP-1i was applied as a control. C, 10 ␮g/ml mannan in sodium carbonate buffer (pH 9.6) soaked overnight. RT-PCR for murine MASP3 mRNA. Forward primers were common to Blocking was done by adding 1% BSA in TBS buffer. Serially diluted MASP1 and MASP3 transcripts, and reverse primers were specific for the murine serum in GVB buffer containing 1% BSA (GVB/BSA) was added L chain of MASP3. RT-PCR was nested using a primer set of 1F/1R for the to mannan-coated wells. After incubation for the indicated periods, the first PCR and another set of 1F/2R for the second PCR (1415 bp). Template wells were washed four times with TBS/Ca/tw buffer. Each well was in- was total RNA derived from each murine liver. To estimate the relative quan- ␮ cubated with diluted purified human C4, at a final concentration of 5 g/ml tities of mRNAs, Gapdh was amplified (729 bp). in GVB/BSA buffer at 37°C for 30 min, and then washed four times with TBS/Ca/tw buffer. The wells were incubated at 37°C for 30 min with FITC-conjugated anti-human C4 Ab (MP Biomedicals), followed by washing wells with TBS/Ca/tw buffer. Fluorescence intensity was detected by Results DTX880 multimode detector (Beckman Coulter) using filters (485 nm for excitation and 535 nm for emission). Integration time was 0.4 s. Generation of MASP1/3 knockout mouse To assess the roles of MASP-1 and MASP-3 in vivo, we estab- C3-deposition on mannan-coated microtiter plates lished a gene targeted mouse that lacks both MASP-1 and Mannan-coated Maxi-Plates, prepared as described above, were blocked in MASP-3. The MASP1/3 gene has 10 exons that encode the H chain TBS/Ca buffer containing 0.1% human serum albumin. Murine serum was common to both MASP1 and MASP3. A targeting vector was con- diluted with BBS buffer (4 mM barbital and 140 mM NaCl (pH 7.4)) structed to replace the second exon with a neomycin resistant gene containing 0.1% human serum albumin, 5 mM CaCl2, and 5 mM MgCl2. Diluted sera were incubated in mannan-coated wells at 37°C. Protein bind- cassette (Fig. 1A). Finally, four neomycin-resistant ES clones ing was stopped by adding 0.2 ml of BBS buffer containing 10 mM EDTA. were obtained, in which the homologous recombination events C3 deposited on well surfaces was detected by anti-human C3c Ab were confirmed by Southern blotting analysis. Two of these were (DakoCytomation) and HRP-conjugated secondary Ab; color was de- independently injected into C57BL/6 blastocysts, and the tected as described above. founder chimeras were bred with C57BL/6J females. Southern Flow cytometry analysis of C3 deposition on zymosan particles blotting analysis of tail DNA from agouti-color pups showed germline transmission of the targeted allele (Fig. 1B). Heterozy- A total of 10% murine sera were incubated with 10 ␮g of zymosan A gous mice (MASP1/3ϩ/Ϫ) were subsequently intercrossed to gen- particles. Reaction was stopped by TBS containing 10 mM EDTA buffer. Ϫ/Ϫ Deposited C3 was stained with anti-human C3c (DakoCytomation), bioti- erate homozygous mice (MASP1/3 ) and, furthermore, back- nated secondary Ab, and FITC-conjugated streptavidin on ice. Acquisitions crossed with C57BL/6J for more than five generations to eliminate were conducted by FACS Calibur (BD Biosciences). the 129/Sv genetic background. The Journal of Immunology 6135

FIGURE 3. C4 deposition activity of murine serum on mannan-coated microtiter wells. C4 deposition assay by murine serum in mannan-coated microtiter wells was measured as described in Materials and Methods. A, F, MASP1/3ϩ/ϩ mouse; E, MASP1/3Ϫ/Ϫ mouse. Fluorescence Intensity of each point shows the average for sera from three mice as relative ﬂuorescence unit (RFU). B, Kinetics of C4 deposition by murine sera in mannan-coated wells. A total of 5 ␮l of serum was reacted in each point. F, MASP1/3ϩ/ϩ mouse; E, MASP1/3Ϫ/Ϫ mouse. rMASP-1K (200 ng) was incubated with MASP1/3Ϫ/Ϫ serum (‚). Fluorescence intencity of each point shows the average for sera from three mice as RFU.

MASP1/3Ϫ/Ϫ mice (from 4 to 10 wk old) were significantly chain coding regions of MASP3 was specifically amplified; how- smaller than control mice ( p Ͻ 0.01, Student t test), although they ever, no such product could be amplified from the liver cDNA of Downloaded from showed no abnormality in either macroscopic appearance or mi- MASP1/3Ϫ/Ϫ mice (Fig. 2C). These results suggest that not only croscopic analysis (data not shown). The average body weights of MASP-1, but also MASP-3, is absent in MASP1/3Ϫ/Ϫ mice. males at 6 wk of age were 20.8 Ϯ 1.97 g (ϩ/ϩ), 21.1 Ϯ 1.34 g (ϩ/Ϫ), and 16.8 Ϯ 1.66 g (Ϫ/Ϫ)(p ϭ 0.0004, compared with ϩ/ϩ) and those of females were 17.9 Ϯ 1.16 g (ϩ/ϩ), 17.5 Ϯ ϩ Ϫ Ϯ Ϫ Ϫ Ͻ 2.38 g ( / ), and 14.8 1.73 g ( / )(p 0.0001, compared http://www.jimmunol.org/ with ϩ/ϩ). The frequency of MASP1/3Ϫ/Ϫ mice developed by intercrossing of MASP1/3ϩ/Ϫ was slightly lower (ϩ/ϩ: ϩ/Ϫ: Ϫ/Ϫϭ 114: 223: 63) than the expected frequency. Although MASP1/3Ϫ/Ϫ mice were fertile, the pups resulting from an intercross of MASP1/ 3Ϫ/Ϫ mice were more vulnerable. A possible reason is that nursing MASP-1- and MASP-3-deficient mothers are too small to provide sufficient milk. Two strains, derived from two independently targeted ES clones, showed the same body weight phenotype and similar frequencies of homozygous pups. This excludes the pos- by guest on September 28, 2021 sibility that another gene locus is additionally disrupted. These data suggest that MASP-1 and/or MASP-3 might be involved, in part, in development and growth.

MASP-1 and MASP-3 are completely absent in MASP1/3Ϫ/Ϫ mice To investigate whether MASP-1 is entirely absent in MASP1/3Ϫ/Ϫ mice, the amount of MASP-1 in mouse serum was estimated by an ELISA system using an anti-mouse MASP-1 Ab. As shown in Fig. 2A, no MASP-1 was detected by this method in the serum of MASP1/3Ϫ/Ϫ mice, whereas MASP-1 in the sera of MASP1/3ϩ/ϩ and MASP1/3ϩ/Ϫ mice was captured in a dose-dependent manner. In another experiment, MBL-MASP complexes in mouse serum were pulled down with mannan-agarose. By Western blotting of the complexes with anti-mouse MASP-1 under nonreducing conditions, both the active form and the proenzyme form of MASP-1 were found in MASP-1-expressing mice (MASP1/3ϩ/ϩ and ϩ/Ϫ MASP1/3 ) (Fig. 2B, lanes 1 and 2, respectively). However, no FIGURE 4. Activation of MASP-2 by mouse serum. A, Individual detectable band derived from MASP-1 was observed in MASP1/ mouse serum derived from MASP1/3ϩ/ϩ (lanes 1 and 2) and MASP1/3Ϫ/Ϫ Ϫ Ϫ 3 / mice (lane 3). Several additional bands (100, 140, and 200 (lanes 3 and 4) was incubated on ice with mannan-agarose in a buffer kDa) were also detected in the serum of MASP1/3ϩ/ϩ. They might containing 1 ␮g/ml aprotinin to prevent activation of MASP-2. Bound be MASP-3 itself or complexes with serine protease inhibitors, MASP-2 was run in SDS-PAGE under reducing conditions and detected by such as C1 esterase inhibitor or ␣2-macrogloblin, although they anti-MASP-2 Ab. B, Effects of MASP-1/3 on MASP-2 activation. Sera Ϫ/Ϫ ϩ/ϩ have not yet been identiﬁed. These results conﬁrmed that MASP-1 from MASP1/3 (lans 1, 3, and 5) and MASP1/3 mice (lanes 2, 4, and Ϫ Ϫ 6) were incubated with mannan-agarose for 10 (lanes 1 and 2), 20 (lanes protein was completely absent from MASP1/3 / mice. Ϫ/Ϫ 3 and 4), and 45 min (lanes 5 and 6), and then subjected to Western blotting To assess whether or not MASP-3 is present in MASP1/3 using an anti-MASP-2 H chain Ab, which recognizes both MASP-2 and mice, RT-PCR of murine MASP3 was conducted, because our at- sMAP. C, Effect of rMASP-1 on MASP-2 activation. Sera from MASP1/ tempt failed to generate a high enough titer of Ab against the L 3ϩ/ϩ (lane 1) and MASP1/3Ϫ/Ϫ mice (lanes 2 and 3), un-supplemented chain of MASP-3. Using liver cDNA from wild-type mouse as a (lanes 1 and 2) or supplemented with rMASP-1n (lane 3), were incubated template, a 1415 bp cDNA fragment spanning both the H and L with mannan-agarose for 10 min. 6136 MASP-1 CAN ACTIVATE THE LECTIN PATHWAY

In the lectin pathway, it has been well known that MASP-2 cleaves C4 and C2. Next, we asked whether MASP-2 was activated in the absence of MASP-1. MBL-MASP complex was captured by mannan-agarose from mouse serum diluted with TBS/Ca buffer, and subsequently subjected to Western blotting using an Ab recognizing the H chain of MASP-2. Aprotinin was able to prevent the activation of MASP-2 completely in MBL complexes derived from sera of MASP1/3ϩ/ϩ and MASP1/3Ϫ/Ϫ (Fig. 4A). The level of Ϫ/Ϫ FIGURE 5. Cleavage of rMASP-2i by rMASP-1K. Ten ␮l of a reaction proenzyme form of MASP-2 (88 kDa) in serum of MASP1/3 ϩ/ϩ containing rMASP-2i (775 ng) was incubated with rMASP-1K (87 ng) or was comparable with that in serum of MASP1/3 , expecting that rMASP-1i (190 ng) for 15, 30, or 60 min, at 37°C. Ten ␮l reaction mixtures the concentrations of MASP-2 in both sera were not different. ϩ ϩ were subjected to Western blotting using an anti-MASP-2 H chain Ab When serum of MASP1/3 / mice was incubated in the absence of under reducing conditions. a: active form of MASP-2i (H chain); p: proen- aprotinin, a band of 62 kDa corresponding active form of MASP-2 zyme form of MASP-2i. was detected, in addition to an 88 kDa band corresponding to the proenzyme form of MASP-2 (Fig. 4B). The bands of ϳ20 kDa seen in all lanes were the murine counterpart of human sMAP, a MASP1/3Ϫ/Ϫ mice show low C4 cleavage ability associated truncated form of MASP-2. In MBL-MASP complex from ϩ ϩ with low MASP-2 activation MASP1/3 / mice, the active form of MASP-2 was observed fol-

Ϫ Ϫ lowing 10, 20, and 45 min of incubation with mannan-agarose Downloaded from To assess the activity of the lectin pathway in MASP1/3 / mice, (Fig. 4B, lanes 2, 4, and 6). In contrast, MASP-1- and MASP-3- we conducted the C4-deposition assay with mannan-coated plates deficient mice revealed no active form of MASP-2 following up to 20 as described in the Materials and Methods. Using sera from ϩ ϩ min of incubation with mannan-agarose (lanes 1 and 3). MASP-2 was MASP1/3 / mice, the amount of C4-deposition on mannan- converted to the activated form after 30 min; however, the amount of coated plates increased in a dose-dependent manner (Fig. 3A). In Ϫ/Ϫ Ϫ/Ϫ active MASP-2 in MASP1/3 mice was lower than that in MASP1/ contrast, C4-deposition from the sera of MASP1/3 mice was ϩ/ϩ Ϫ/Ϫ ϩ ϩ 3 mice. Addition of rMASP-1n into the sera of MASP1/3 mice http://www.jimmunol.org/ significantly lower than that in MASP1/3 / mice. Kinetics plots restored the MASP-2 activation (Fig. 4C). of C4-deposition using 5 ␮l of serum also showed a very low MASP1/3Ϫ/Ϫ B activity in mice (Fig. 3 ). These results support the Cleavage of recombinant inactive MASP-2 mutant (rMASP-2I) possibility that MASP-1 or MASP-3 is involved in the activation by rMASP-1K of the lectin complement pathway. To determine whether lack of MASP-1 contributes to the reduced activity of complement acti- To assess whether MASP-1 cleaves MASP-2 directly, we incu- vation in MASP1/3Ϫ/Ϫ mice, recombinant mouse MASP-1K was bated a recombinant mutant of MASP-2, rMASP-2i, with rMASP- added to the serum. rMASP-1K was modified from native-type of 1K. Previous studies have shown that rMASP-2 was easily autoactivated during its preparation (13). Therefore, we used

MASP-1 by replacing the arginine residue at the reactive site P1 by guest on September 28, 2021 for activation of MASP-1 with lysine. The same strategy was con- rMASP-2i as a ligand. This mutant was not cleaved by itself be- ducted when recombinant MASP-2 was expressed by Chen and cause it lacks protease activity. As shown in Fig. 5, rMASP-1K Wallis (22). Their mutant MASP-2K reduced the rate of its auto- was able to cleave rMASP-2i directly. In contrast, rMASP-1i could activation during synthesis and purification. We have attempted to not cleave rMASP-2i, even if the concentration of rMASP-1i was produce recombinant native-type of mouse MASP-1 by baculovi- more than 2-fold higher than that of rMASP-1K, indicating the rus expression system, but very poor protein could be purified. specific activity of rMASP-1K.

Instead of the native type of MASP-1, a large amount of Ϫ/Ϫ rMASP-1K could be puriﬁed from a high ﬁve cell line that was MASP1/3 mice show low C3 cleavage ability stably transfected with an expression vector. The C4-deposition To assess C3 activation by the lectin pathway in mouse serum, activity of MASP1/3Ϫ/Ϫ mouse serum was restored by the recon- diluted serum was incubated with mannan-coated plates, and de- stitution of rMASP-1K (Fig. 3B). posited endogenous C3 was determined using an anti-C3c Ab. C3

FIGURE 6. C3 deposition activity of murine serum on mannan-coated microtiter wells. C3 deposition assay in mannan-coated microtiter wells by mouse serum. A, Diluted serum was reacted in mannan-coated wells at 37°C for 30 min. Bound C3 was detected by HRP-conjugated anti-C3c and 3,3Ј,5,5Ј- tetrametylbenzidine. F and E, MASP1/3ϩ/ϩ mouse and MASP1/3Ϫ/Ϫ mouse, respectively. The absorbance of each point shows the average for sera from three mice. B, Kinetics of C3 deposition by murine sera on mannan-coated wells. A total of 2 ␮l of serum was reacted for the indicated time. F and E, MASP1/3ϩ/ϩ mouse and MASP1/3Ϫ/Ϫ mouse, respectively. rMASP-1K (200 ng) was incubated with MASP1/3Ϫ/Ϫ serum (‚). The absorbance of each point shows the average for sera from three mice. The Journal of Immunology 6137

deposition from the sera of MASP1/3Ϫ/Ϫ mice was much lower than C3 deposition from the sera of MASP1/3ϩ/ϩ mice (Fig. 6A). The kinetics of C3 activation by 2 ␮l of serum was very slow in MASP1/3Ϫ/Ϫ mice (Fig. 6B). By adding rMASP-1K into the serum of MASP1/3Ϫ/Ϫ mice, the C3-deposition activity was restored. We also examined C3 activation by yeast-derived-zymosan particles. When zymosan particles were incubated with normal murine serum, the signiﬁcant C3 deposition on the particles was observed for 1-min incubation (Fig. 7A). This activity was almost inhibited by adding 0.1 M mannose. Further incubation (5 min) allowed the C3 deposition on the particles by normal murine serum even if adding mannose (Fig. 7B). These results suggested that the lectin pathway plays an important role in the early event for complement activation on zymosan surface, although alternative pathway might be mainly involved in the late complement activation. In MASP1/3Ϫ/Ϫ serum, there was no C3 deposition on zymosan particles for 1-min incubation (Fig. 7C). rMASP-1n, but not rMASP-1i, could increase the C3 deposition activity in serum of Ϫ Ϫ MASP1/3 / for 1-min incubation (Fig. 7D). Downloaded from

Discussion We have generated mice deﬁcient in MASP-1 and MASP-3 by gene targeting, and have used these mice to investigate the role of MASP-1 in the activation of the lectin complement pathway.

Among MASP-1, MASP-2, and MASP-3, only MASP-2 has C4 http://www.jimmunol.org/ cleavage activity (8, 12). A number of studies in vitro supported the possibility that MASP-2 alone is sufficient to activate complement in the absence of MASP-1 and MASP-3. Surprisingly, however, serum derived from MASP1/3Ϫ/Ϫ has a decreased ability to activate C4 on mannan-coated microtiter wells. No activation of MASP-2 was observed in MASP-1- and MASP-3-deficient serum, when serum was incubated with mannan-agarose for 10 min, whereas activation of MASP-2 was observed in MASP-1- and MASP-3-containing serum (Fig. 4). rMASP-1K restored C4 and by guest on September 28, 2021 MASP-2 activation in MASP-1- and MASP-3-deficient serum (Figs. 3 and 4). These results support the possibility that MASP-1 also contributes to the activation of the lectin pathway at the step of MASP-2 activation. The precise mechanism by which MASP-1 affects the activation of MASP-2 is not clear. One possible explanation is that MASP-1 cleaves MASP-2 directly. To verify this possibility, we incubated a mutant of rMASP-2i with rMASP-1K. As shown in Fig. 5, it was confirmed that rMASP-1K possesses activity to cleave rMASP-2i in vitro. Another possibility is that MASP-1 enhances the autoactivation of MASP-2. In any case, it has been previously reported that the complex of MASP-1 contains MBL and sMAP, but not MASP-2, using an immunoprecipitation assay with an anti-MASP-1 Ab (23). So far, there is no evidence that MASP-1 and MASP-2 associate with each other in an oligomer of MBL. In peripheral blood, MBL may form individual complexes with MASP-1, MASP-2, or MASP-3, respectively. When these MBL/MASPs complexes bind to carbohydrates on microorganisms, it is possible that in the vicinity of this binding, MBL/MASP-1 may interact with MBL/MASP-2. MASP-2 was activated even in the absence of MASP-1 and MASP-3, when the serum was incubated with mannan for Ͼ30 min (Fig. 4). It has been reported that rMASP-2 is easily autoactivated and rMASP-2 alone associated with MBL is sufficient for FIGURE 7. Flow cytometric analysis of C3 deposition on zymosan. Murine sera (10%) were incubated with yeast-derived-zymosan particles at 37°C. Deposited C3 on the particles was detected by anti-human C3c Ab, adding 0.1 M mannose. C, Comparison of C3 deposition activity between biotinated secondary Ab, and FITC-streptavidin. A and B, C57BL/6 murine serum of C57BL/6 (opened histogram) and that of MASP1/3Ϫ/Ϫ (filled serum was incubated for 1 (A) and 5 min (B) (gray filled histogram). As histogram). D, MASP1/3Ϫ/Ϫ serum (grayed histogram) was reconstituted negative controls, solid line histograms indicate that reaction was imme- with 100 ng of rMASP-1n (solid line) and 300 ng of rMASP-1i (mutant diately stopped by EDTA. Dot line histograms represent the inhibition by type) (dot line). Incubation time was 1 min. 6138 MASP-1 CAN ACTIVATE THE LECTIN PATHWAY the activation of C4 (13). However, we would like to point out the 4. Brouwer, N., K. M. Dolman, R. van Zwieten, E. Nieuwenhuys, M. Hart, fact that MASP-2 circulates as a zymogen in peripheral blood, L. A. Aarden, D. Roos, and T. W. Kuijpers. 2006. Mannan-binding lectin (MBL)- mediated opsonization is enhanced by the alternative pathway amplification loop. suggesting that some unknown mechanism prevents autoactivation Mol. Immunol. 43: 2051–2060. of MASP-2. A possible explanation is that a physiological con- 5. Kawasaki, T., R. Etoh, and I. Yamashina. 1978. Isolation and characterization of a mannan-binding protein from rabbit liver. Biochem. Biophys. Res. Commun. 81: formation of MBL that binds to MASP-2 or some inhibitors (C1 1018–1024. inhibitor or ␣2-M) may prevent the autoactivation of MASP-2. 6. Matsushita, M., Y. Endo, S. Taira, Y. Sato, T. Fujita, N. Ichikawa, M. Nakata, When MBL recognizes a carbohydrate structure, conformational and T. Mizuochi. 1996. A novel human serum lectin with collagen- and fibrin- ogen-like domains that functions as an opsonin. J. Biol. Chem. 271: 2448–2454. changes in MBL structure may alter the stability of MASP-2, as 7. Matsushita, M., and T. Fujita. 1992. Activation of the classical complement path- reported by Wallis (24). Under physiological conditions, MASP-1 way by mannose-binding protein in association with a novel C1s-like serine may affect the structure or stability of the MBL-MASP-2 complex, protease. J. Exp. Med. 176: 1497–1502. 8. Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen, thereby resulting in activation of MASP-2. K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. Holmskov, et al. 1997. A We also investigated the deposition of C3 from murine serum second serine protease associated with mannan-binding lectin that activates complement. Nature 386: 506–510. onto mannan. All complement pathways contribute C3 cleavage to 9. Dahl, M. R., S. Thiel, M. Matsushita, T. Fujita, A. C. Willis, T. Christensen, form C3b and C3a that have important roles in the proceeding T. Vorup-Jensen, and J. C. Jensenius. 2001. MASP-3 and its association with complement cascades. In MASP-1- and MASP-3-deficient serum, distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15: 127–135. a significantly lower amount of C3 deposition was observed than 10. Takahashi, M., Y. Endo, T. Fujita, and M. Matsushita. 1999. A truncated form of that in MASP-1- and MASP-3-containing serum. No significant mannose-binding lectin-associated serine protease (MASP)-2 expressed by alter- difference of concentrations of C3 and C4 between wild-type and native polyadenylation is a component of the lectin complement pathway. Int. Immunol. 11: 859–863. MASP-1/3-deficient sera was observed (data not shown). 11. Stover, C. M., S. Thiel, M. Thelen, N. J. Lynch, T. Vorup-Jensen, J. C. Jensenius, Downloaded from MASP-1- and MASP-3-deficient serum has decreased activity to and W. J. Schwaeble. 1999. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single form a C3 convertase, C4b2a, supporting that the possibility that structural gene. J. Immunol. 162: 3481–3490. C3 deposition from MASP-1- and MASP-3-deficient serum was 12. Matsushita, M., S. Thiel, J. C. Jensenius, I. Terai, and T. Fujita. 2000. Proteolytic also lower. From several reports, it is clear that MASP-1 can activities of two types of mannose-binding lectin-associated serine protease. J. Immunol. 165: 2637–2642. cleave C2 (12, 22). In addition to MASP-2, therefore, MASP-1 13. Vorup-Jensen, T., S. V. Petersen, A. G. Hansen, K. Poulsen, W. Schwaeble, activates C2 and also plays an important role in the formation of R. B. Sim, K. B. Reid, S. J. Davis, S. Thiel, and J. C. Jensenius. 2000. Distinct http://www.jimmunol.org/ the C3 convertase, C4b2a. The other possible explanation is that pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine pro- MASP-1 directly activates C3, as reported previously in human, tease-2. J. Immunol. 165: 2093–2100. although this C3 cleaving activity is questioned (15, 16). We now 14. Matsushita, M., and T. Fujita. 1995. Cleavage of the third component of complement (C3) by mannose-binding protein-associated serine protease (MASP) have mice deficient for all MASP proteins in our laboratory, and with subsequent complement activation. Immunobiology 194: 443–448. this question is currently under investigation. 15. Rossi, V., S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius, and G. J. Arlaud. MASP1 and MASP3 mRNA were transcripted from same gene 2001. Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2. J. Biol. Chem. 276: 40880–40887. by alternative splicing. A common exon between them has been 16. Ambrus, G., P. Gal, M. Kojima, K. Szilagyi, J. Balczer, J. Antal, L. Graf, targeted for disruption to make the knockout mice. It has been A. Laich, B. E. Moffatt, W. Schwaeble, et al. 2003. Natural substrates and in- shown that MASP-3 was also absent in these mice. Therefore, the hibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on by guest on September 28, 2021 recombinant catalytic fragments. J. Immunol. 170: 1374–1382. Ϫ/Ϫ affect of deficiency of MASP-3 in MASP1/3 could not be elim- 17. Ihara, I., H. Ueda, A. Suzuki, and M. Kawakami. 1982. Physicochemical prop- inated. However, Dahl et al. (9) has reported that MASP-3 inhib- erties of a new bactericidal factor, Ra-reactive factor. Biochem. Biophys. Res. Commun. 107: 1185–1190. ited the activation of C4 and MASP-2, suggesting that MASP-3 18. Takayama, Y., F. Takada, A. Takahashi, and M. Kawakami. 1994. A 100-kDa may negatively regulate the lectin pathway. Furthermore, protein in the C4-activating component of Ra-reactive factor is a new serine rMASP-1K could completely restore the activation of C4 and C3 protease having module organization similar to C1r and C1s. J. Immunol. 152: Ϫ/Ϫ 2308–2316. in MASP1/3 . All together, at least positive effects of MASP-1 19. Endo, Y., M. Takahashi, M. Nakao, H. Saiga, H. Sekine, M. Matsushita, in the lectin pathway can be concluded from our study, although M. Nonaka, and T. Fujita. 1998. Two lineages of mannose-binding lectin-asso- effect of MASP-3 should be discussed in future study. ciated serine protease (MASP) in vertebrates. J. Immunol. 161: 4924–4930. 20. Stover, C. M., N. J. Lynch, M. R. Dahl, S. Hanson, M. Takahashi, In conclusion, we generated MASP-1- and MASP-3-deficient M. Frankenberger, L. Ziegler-Heitbrock, I. Eperon, S. Thiel, and mice, and found that MASP-1 plays a pivotal role in the activation W. J. Schwaeble. 2003. Murine serine proteases MASP-1 and MASP-3, components of the lectin pathway activation complex of complement, are encoded by a of the lectin pathway, probably through activation of MASP-2. single structural gene. Genes Immun. 4: 374–384. 21. Iwaki, D., and T. Fujita. 2005. Production and purification of recombinants of mouse MASP-2 and sMAP. J. Endotoxin. Res. 11: 47–50. Disclosures 22. Chen, C. B., and R. Wallis. 2004. Two mechanisms for mannose-binding protein The authors have no financial conflict of interest. modulation of the activity of its associated serine proteases. J. Biol. Chem. 279: 26058–26065. 23. Thiel, S., S. V. Petersen, T. Vorup-Jensen, M. Matsushita, T. Fujita, C. M. Stover, References W. J. Schwaeble, and J. C. Jensenius. 2000. Interaction of C1q and mannan- 1. Walport, M. J. 2001. Complement: second of two parts. N. Engl. J. Med. 344: binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, 1140–1144. and the MBL-associated protein MAp19. J. 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