Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway

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Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway

This information is current as Manabu Hayashi, Takeshi Machida, Yumi Ishida, Yusuke of September 27, 2021. Ogata, Tomoko Omori, Mika Takasumi, Yuichi Endo, Toshiyuki Suzuki, Masayuki Sekimata, Yoshimi Homma, Masahito Ikawa, Hiromasa Ohira, Teizo Fujita and Hideharu Sekine

J Immunol published online 9 August 2019 Downloaded from http://www.jimmunol.org/content/early/2019/08/09/jimmun ol.1900605

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 9, 2019, doi:10.4049/jimmunol.1900605

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway Manabu Hayashi,*,† Takeshi Machida,* Yumi Ishida,* Yusuke Ogata,* Tomoko Omori,* Mika Takasumi,*,† Yuichi Endo,* Toshiyuki Suzuki,‡ ‡ x { † Masayuki Sekimata,‖ Yoshimi Homma, Masahito Ikawa, Hiromasa Ohira, Teizo Fujita, and Hideharu Sekine* The complement system, a part of the innate immune PRMs of the LP form a complex with MBL-associated serine system, can be activated via three different pathways. proteases (MASPs) (2). Once PRMs bind to their activators or In the alternative pathway, a factor D (FD) plays es- ligands, the serine proteases complexed therewith are activated Downloaded from sential roles in both the initiation and the amplification and cleave C4 and C2 to form a C3 convertase, C4b2a. loop and circulates as an active form. Mannose-binding Unlike the CP and LP, activation of the AP is initiated by lectin–associated serine proteases (MASPs) are key en- spontaneous hydrolysis of C3 at low levels. Once C3(H2O) zymes of the lectin pathway, and MASP-1 and/or binds to complement factor B (FB), complement factor D MASP-3 are reported to be involved in the activation (FD), circulating as an active form cleaves FB to form an http://www.jimmunol.org/ of FD. In the current study, we generated mice mono- initial C3 convertase, C3(H2O)Bb. C3(H2O)Bb cleaves C3 specifically deficient for MASP-1 or MASP-3 and to C3a and C3b; the latter covalently binds to microbial found that the sera of the MASP-1–deficient mice lacked surfaces and binds FB, which will be cleaved by FD to form lectin pathway activity, but those of the MASP-3–deficient the AP C3 convertase, C3bBb. Both the C3 convertases mice lacked alternative pathway activity with a zymogen (i.e., C4b2a for the CP and LP, and C3bBb for the AP) cleave FD. Furthermore, the results indicate that MASP-3 but C3 to C3a and C3b; the latter binds FB, which is again not MASP-1 activates the zymogen FD under physiolog- cleaved by FD to form the additional C3bBb, generating large ical conditions and MASP-3 circulates predominantly as amounts of C3b via the amplification loop. Therefore, FD by guest on September 27, 2021 an active form. Therefore, our study illustrates that, in plays essential roles in the initiation of the AP and the sub- sequent amplification loop that significantly contributes to mice, MASP-3 orchestrates the overall complement re- immunological responses elicited by all complement activa- action through the activation of FD. The Journal of tion pathways (1). Immunology, 2019, 203: 000–000. MASP-1 and MASP-3 are produced mainly in the liver by alternative splicing from the common Masp1 gene, with the he complement system plays important roles in innate result that they have a common H chain and distinct L chains immunity, maintaining biological homeostasis and (2). The L chain consists of the serine protease domain, T acting as immune surveillance (1). The complement transcribed from either MASP-1–specific split exons or an system can be activated via three different pathways; the MASP-3–specific single exon. Previously, we generated a classical pathway (CP), the lectin pathway (LP), and the alter- MASP-1/3–deficient mouse by targeting the common exon of native pathway (AP) (2). Activation of the CP and LP are ini- Masp1 (3, 4). These mice showed a lack of AP activity with a tiated when pattern recognition molecules (PRMs) (i.e., C1q in zymogen FD (pro-FD) in addition to a lack of LP activity, the CP; mannose-binding lectin (MBL), ficolins, and collectins indicating that MASP-1 and/or MASP-3 are required for in the LP) in a complex with serine proteases bind to ligands. activation of the AP. To elucidate the individual roles of

*Department of Immunology, Fukushima Medical University School of Medicine, Fukushima Address correspondence and reprint requests to Dr. Takeshi Machida, Department of City, Fukushima 960-1295, Japan; †Department of Gastroenterology, Fukushima Medical Immunology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, University School of Medicine, Fukushima City, Fukushima 960-1295, Japan; ‡Radioisotope Fukushima 960-1295, Japan. E-mail address: [email protected] Research Center, Fukushima Medical University School of Medicine, Fukushima City, x The online version of this article contains supplemental material. Fukushima 960-1295, Japan; Department of Biomolecular Science, Fukushima Medical Uni- { versity School of Medicine, Fukushima City, Fukushima 960-1295, Japan; Research Institute Abbreviations used in this article: AP, alternative pathway; CP, classical pathway; FB, ‖ for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; and Fukushima factor B; FD, factor D; gRNA, guide RNA; LP, lectin pathway; MASP, MBL-associated Prefectural General Hygiene Institute, Fukushima City, Fukushima 960-8141, Japan serine protease; MBL, mannose-binding lectin; PRM, pattern recognition molecule; pro-FD, zymogen FD; WT, wild-type. ORCIDs: 0000-0002-2213-3918 (M.H.); 0000-0002-8346-3099 (T.M.); 0000-0003- 3069-2303 (M.S.); 0000-0001-9859-6217 (M.I.); 0000-0002-5311-7566 (H.S.). Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 Received for publication June 6, 2019. Accepted for publication July 17, 2019. This work was supported by Grant-in-Aid for Scientiﬁc Research (C) JP19K07610 and by Grant-in-Aid for Early-Career Scientists JP18K15789 from the Japan Society for the Promotion of Science.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900605 2 CUTTING EDGE: ROLE OF MASP-3 IN COMPLEMENT SYSTEM

MASP-1 and MASP-3 in the activation of the LP and of San Diego, CA), MBL-A (Hycult Biotech, Plymouth Meeting, PA), and the AP via the activation of FD, we generated mice mono- MBL-C (Hycult) were measured using ELISA kits according to the manu- facturers’ instructions. specifically deficient for MASP-1 or MASP-3. We found that MASP-1 and MASP-3 play independent roles in the physi- C4 and C3 deposition assays and hemolytic assay ological activation of the LP and AP, respectively. LP activity was measured by a C4 deposition assay as previously described (3). AP activity was measured by C3 deposition assays using microplates coated Materials and Methods with zymosan, mannan, or LPSs derived from Escherichia coli, Salmonella Generation of mice monospecifically deficient for MASP-1 or MASP-3 typhimurium, and Pseudomonas aeruginosa (Sigma-Aldrich) in 0.1 M sodium carbonate buffer (pH 9.6) by overnight incubation at 4˚C. C3 deposition Mice monospecifically deficient for MASP-1 or MASP-3 were generated on the microplates was assayed as described previously (4). C3 deposition using the CRISPR/Cas9 system. Guide RNAs (gRNAs) for the deletion of on zymosan was also assayed by flow cytometry as described previously (3). MASP-1–specific exons (gRNA no. 1, 59-TACCCATTGGGGTTACCAGC-39 C3 deposition levels were expressed as mean fluorescence intensity obtained and gRNA no. 2, 59-CGCTTCTCTTACCATTTGGT-39) or for the deletion of by BD FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). a MASP-3–specific exon (gRNA no. 3, 59-TGGCTAAGTCGCTTTTGGAC-39 The hemolytic assay against rabbit erythrocyte was performed as described and gRNA no. 4, 59-GCATCTTCGACTAATGCCTG-39) were designed using previously (4). the CRISPRdirect Web software (5). The annealed oligonucleotides were inserted into the BbsI restriction site in the pSpCas9(BB)-2A-Puro (PX459) V2.0 Western blotting (Addgene, Watertown, MA). These plasmids were microinjected into zygotes isolated from C57BL/6J mice, then, the injected zygotes were transferred to Western blotting of MASPs was performed using mouse sera, plasma, and pseudopregnant female mice. The pups were backcrossed with C57BL/6J serum MBL–MASP complex purified with mannan-agarose (Sigma-Aldrich). mice (also used as wild-type [WT] mice; CLEA Japan, Tokyo, Japan) to Serum and plasma samples were prepared in the presence of a protease in- Downloaded from yield heterozygous F1 mutant mice, followed by inbreeding to yield hetero- hibitor mixture (Sigma-Aldrich) and pretreated with Proteome Purify 2 2 2 2 2 2 2 zygous (Masp1+/ or Masp3+/ ) and homozygous (Masp1 / or Masp3 / ) Mouse Serum Protein Immunodepletion Resin (R&D Systems, Minneapolis, mutant mice. Genotyping PCR was performed using their tail genomic DNAs MN). Western blotting of FD was performed as described previously (4). with specific primers as follows: for genotyping of the MASP-1–specific exons, m1F After SDS-PAGE, target proteins were analyzed by Western blotting using (59-GGATGGTTTCTAAGGGCATTC-39), m1R-1 (59-GTCTTGGGGCCTAC- rabbit anti-mouse MASP-3 L chain Ab or rabbit anti-mouse FD Ab, followed TAGTC-39), and m1R-2 (59-AACACAGTGTTCTCAGTTTGG-39)wereusedto by incubation with HRP-conjugated swine anti-rabbit Ig Ab (Dako, Glostrup, amplify 1275- and 935-bp fragments for the WT and mutant alleles, respectively. Denmark).

For genotyping of the MASP-3–specific exon, m3F (59-CCCAGTCTGCCAT- http://www.jimmunol.org/ GAAATCT-39), m3R-1 (59-CACCACTCCCTCTGTGGATT-39), and m3R-2 Statistical analysis (59-CCAGGTGAGAAGAATCAGCAG-39) were used to amplify 588- and Statistical analyses were performed by Dunnett multiple comparison test or 500-bp fragments for the WT and mutant alleles, respectively. DNA sequencing two-way ANOVA using GraphPad Prism 8.0 software (GraphPad Software, was performed to confirm the editing of the genome with deletion of targeted San Diego, CA). Differences with a p value , 0.05 were considered statis- exon(s) by Macrogen (Tokyo, Japan). tically significant. MASP-2–deficient (6) and MASP-1/3–deficient mice (3) have previously been described. All animal experiments that included housing, breeding, and use of the mice were reviewed and approved by the Animal Experiments Results and Discussion Committee of Fukushima Medical University (approval no. 29032) and Generation of mice monospecifically deficient for MASP-1 or MASP-3 conducted in accordance with the guidelines for the care and use of laboratory

using the CRISPR/Cas9 system by guest on September 27, 2021 animals established by the Committee. We generated mice monospecifically deficient for MASP-1 2 2 2 2 Real-time RT-PCR (Masp1 / )orMASP-3(Masp3 / )bydeletionofthe Total RNAs were extracted from mouse liver using an illustra RNAspin Mini Masp1 gene exons 13–18 or an exon 12 encoding the L chain Isolation Kit (GE Healthcare, Buckinghamshire, U.K.) and used for synthesis of MASP-1 or MASP-3, respectively (Fig. 1A). Deletion of of first-strand cDNA using the Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA). Real-time PCR was performed using the synthesized mouse liver targeted exon(s) was confirmed by PCR analysis of mouse tail cDNA, Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, genomic DNA (Fig. 1B) and sequencing of the PCR products MA), and the StepOne Real-Time PCR System (Applied Biosystems, Foster (Supplemental Fig. 1A). Selective deficiency of MASP-1 or 9 City,CA).Primersetsusedforreal-timePCRareasfollows:5-AGTGCTCAA- MASP-3 in mice induced by genome editing was confirmed GAGAAGCCTGC-39 and 59-AGCAGCTGTCAAAACCCAGT-39 for MASP-1; 59-AGTGCTCAAGAGAAGCCTGC-39 and 59-ACCCTCGATGTGTCTTC- by measuring their hepatic mRNA levels and serum levels of CAC-39 for MASP-3; and 59-CAAAGACCAAGTGCTCGTCA-39 and 59-CT- MASPs in a complex with MBL (Supplemental Fig. 1B, 1C). 2 2 TCTCCAATTCGATCTCGC-39 for MAp44, a splice variant of Masp1 gene. Of interest, hepatic mRNA levels of MASP-1 in the Masp3 / b-actin cDNA was amplified using specific primers (59-CTTTGCAGCTCCTT- CGTTGC-39 and 59-ATTCCCACCATCACACCCTG-39)asaninternalcontrol mice were significantly higher than those in the WT mice for normalizing mRNA levels among the samples. (p , 0.0001), whereas serum levels of MASP-3 were signif- 2 2 icantly higher in the Masp1 / mice than in the WT mice Ab preparation (p = 0.0001). In addition, there were no significant differences The anti-mouse MASP-1 L chain polyclonal Ab was raised in the MASP-1– in hepatic mRNA levels of MAp44, a splice variant of the deficient mice. The anti-mouse MASP-3 L chain polyclonal Ab was raised in a Masp1 gene lacking a serine protease domain (9). We further rabbit. A mouse MASP-1 C-terminal peptide (NKDWIQRITGVRN) and a mouse MASP-3 C-terminal peptide (CLWEEMNSPRAVRDLQVER) were measured the serum levels of complement C3, C4, MBL-A, synthesized by Scrum (Tokyo, Japan), and Immuno-Biological Laboratories MBL-C, and ficolin-A, and there were no significant differ- (Gunma, Japan), respectively. ences between the four groups (Supplemental Fig. 1D). These ELISA results indicate that the impact of the absence of MASP-1 and/or MASP-3 on serum levels of C3, C4, MBL-A, MBL-C, Serum levels of MASP-1 and MASP-3 in a complex with MBL were measured by ELISA using microplates coated with 10 mg/well mannan (Sigma-Aldrich, and ficolin-A is minimal. St. Louis, MO) in 0.1 M sodium carbonate buffer (pH 9.6). Mouse serum Independent roles of MASP-1 and MASP-3 in the activation of the was diluted at 10% with TBS (pH 7.4) containing 5 mM CaCl2, and the captured MASP-1 or MASP-3 was detected with mouse anti-mouse MASP-1 complement pathways L chain Ab or rabbit anti-mouse MASP-3 L chain Ab and appropriate To define the roles of MASP-1 and MASP-3 in complement HRP-conjugated secondary Abs. Serum levels of C3 (7), ficolin-A (8), total FD, and pro-FD (4), were activation, we evaluated the LP activity in sera from the mice measured by ELISA as described previously. Serum levels of C4 (MyBioSource, deficient for MASP-1 or MASP-3 by the C4 deposition assay. The Journal of Immunology 3

FIGURE 1. Generation of mice monospecifically deficient for MASP-1 or MASP-3. (A) A scheme of mouse Masp1 gene that consists of exons 1–11 encoding the common H chain between MASP-1 and MASP-3, an exon 12 encoding the MASP-3 L chain, and exons 13–18 encoding the MASP-1 L chain. Thin vertical arrows indicate the location of two sets of gRNAs targeting introns for deletion of MASP-1– or MASP-3–specific exon(s). Bold horizontal arrows indicate the 2/2 2/2 location of the primers used for genotyping. (B) Genotyping of Masp1 (left panel) or Masp3 mice (right panel) using mouse tail genomic DNA. M, Downloaded from 2 2 2 molecular size marker; +/+, WT; +/ , heterozygous mutant; / , homozygous mutant.

Sera from the MASP-1–deficient mice showed little to no C4 deposition (Fig. 2B). Also, kinetic analysis of the C3 depo- deposition, whereas sera from the WT and MASP-3–deficient sition on zymosan particles by flow cytometry showed little to mice showed obvious C4 deposition with significant differ- no C3 deposition on zymosan in sera from the MASP-3– ences between the groups (p = 0.0027) (Fig. 2A). These re- deficient mice (Fig. 2C, Supplemental Fig. 2A). By contrast, http://www.jimmunol.org/ sults indicate that MASP-1 plays an essential role in the sera from the WT and MASP-1–deficient mice showed ap- physiological LP activation and is consistent with the previous parent C3 deposition on zymosan time dependently. In ad- results (3). dition, a rabbit erythrocyte hemolytic assay demonstrated no We next evaluated the AP activity in sera from the mice. Sera hemolysis in sera from the MASP-3–deficient mice (Fig. 2D). from the MASP-3–deficient mice failed to deposit C3 on Under physiological conditions, therefore, it is clear that microplates coated with zymosan, mannan, or LPSs, whereas MASP-3 plays an essential role in the AP activation, whereas sera from the WT and MASP-1–deficient mice showed C3 MASP-1 is dispensable for the AP activation in mice. Our by guest on September 27, 2021

FIGURE 2. MASP-1 contributes to the LP activation, whereas MASP-3 contributes to the AP activation. (A) C4 deposition activity of mouse serum on mannan- coated microtiter wells (n = 3–5). (B) C3 deposition activity of mouse serum on microtiter wells coated with zymosan, mannan, or LPSs from three different microorganisms (n = 3–6). (C) Kinetic analysis of C3 deposition activity of mouse serum on zymosan particles by flow cytometry (n = 3). (D) Serum hemolytic activity using rabbit erythrocytes (n = 3). (E) The activation state of circulating FD analyzed by Western blotting. Data are represented as means 6 SEM. Asterisks represent statistical differences against the values for the WT mice at *p , 0.05, **p , 0.01, ***p , 0.001 by Dunnett multiple comparisons at each point of age and serum concentration for (C) and (D), respectively. 4 CUTTING EDGE: ROLE OF MASP-3 IN COMPLEMENT SYSTEM results differ from previous in vitro studies that have shown MASP-3 circulates predominantly as an active form in WT MASP-1 to be essential for LPS-induced but not for zymosan- and MASP-1–deficient mice (Fig. 3B). We further analyzed induced AP activation (10). In this study, sera from the the activation state of serum MASP-3 in a complex with MBL MASP-1–deficient mice did not show altered LPS-induced in the circulation. As shown in Fig. 3C, MASP-3, which was AP activity compared with that from the WT mice (Fig. 2B). collected with mannan-agarose from WT and MASP-1– Thus, it is unlikely that MASP-1 plays a physiological role in deficient sera in the presence of protease inhibitors, was also AP activation induced by LPS. activated. Taken together, these data indicate that MASP-3, FD circulates predominantly as an active form and plays an including that in a complex with MBL, circulates as an essential role in the activation of the AP (11). We previ- active form. ously reported that FD circulated as a pro-FD in MASP-1/3– Our previous studies showed mice deficient for MASP-1/3 deficient mice that lack AP activity (4). However, it remained had a pro-FD in the circulation and lacked AP activity in uncertain whether MASP-1, MASP-3, or both are required addition to lacking LP activity (3, 4). Although it has been for the activation of FD. In this study, FD was detected demonstrated that MASP-1 has broad catalytic activity against predominantly as a pro-FD in the sera of the MASP-3– components of the AP, including C3, FD, and MASP-3 deficient mice, although it was detected as an active form, (2, 4), our studies evaluating AP activity in MASP-1– which lacks five N-terminal amino acids of pro-FD (4), in deficient sera did not show attenuation of AP activity but, the sera of the WT and MASP-1–deficient mice (Fig. 2E, rather, enhanced AP activity compared with that of WT sera,

Supplemental Fig. 2B). Although both MASP-1 and MASP-3 likely because of elevated MASP-3 levels in the sera of the Downloaded from can cleave pro-FD in in vitro experiments (4, 12, 13), our MASP-1–deficient mice (Supplemental Fig. 1C). In addition, in vivo results clearly indicate that MASP-3 is essential for the no alteration in the morphology of circulating FD was observed in activation of FD. This study, using mice monospecifically de- the MASP-1–deficient mice. In contrast, MASP-3–deficient sera ficient for MASP-1 or MASP-3, is, to our knowledge, the first showed a lack of AP activity with pro-FD. As summarized in to report the independent roles of MASP-1 and MASP-3 in the Fig. 4, MASP-1 and MASP-3 are required for the activation of physiological activation of the LP and AP via FD activation. MASP-2 and FD, respectively. Thus, in vivo, MASP-1 and http://www.jimmunol.org/ MASP-3 play independent roles in the activation of the LP MASP-3 circulated as an active form regardless of the role of MASP-1 and AP. MASP-1 circulates as an inactive or proenzyme form, and The complement system plays an important role in host it turns into an active form when the PRM/MASP-1 com- defense. However, it is also involved in the development of plexes bind to carbohydrates (2). Recently, Oroszlań et al. (14) numerous inflammatory disorders, including autoimmune demonstrated that MASP-3 circulates as an active form in diseases. We previously reported the essential roles of MASP-1 humans and proposed a mechanism for MASP-3 activation and/or MASP-3 in the development of murine models of

by MASP-1. However, it is unknown whether MASP-3 cir- Ab-induced rheumatoid arthritis or systemic lupus eryth- by guest on September 27, 2021 culates as an active form in the absence of MASP-1 or how ematosus (lupus) (7, 16). Although the activation of the MASP-3 is activated in vivo. To elucidate the activation state complement system in both models seems to be initiated via of circulating MASP-3 and its interaction with MASP-1, we the CP, the data demonstrated significant contributions of analyzed the activation state of MASP-3 by Western blotting the LP and/or AP to the development of immune complex– using mouse MASP-3 L chain–specific Ab. mediated arthritis or glomerulonephritis. The reduction in Strikingly, MASP-3 was detected predominantly as an active glomerular injury observed in MASP-1/3–deficient lupus- form in the sera of the MASP-1–deficient and MASP-2– prone MRL/lpr mice is more pronounced than in MRL/lpr deficient mice as well as in the sera of the WT mice (Fig. 3A, mice deficient for FD or FB, suggesting that there is an ad- Supplemental Fig. 3). We also analyzed MASP-3 in plasma ditive beneficial effect of the absence of the LP activity in because MASP-1 and MASP-2 in a complex with MBL can addition to the AP activity to protect from glomerular injury. potentially be activated by blood coagulation (15). Similarly, Recent studies suggest a novel role of MASP-1 in the blood MASP-3 was detected as an active form in plasma of the coagulation system (17), a disorder which can also be involved WT and MASP-1–deficient mice. These data indicate that in inflammatory vascular diseases, such as lupus. Further studies

FIGURE 3. The activation state of circulating MASP-3 analyzed by Western blotting. (A) Serum MASP-3. The lower arrow indicates the L chain of cleaved MASP-3, and the upper arrow indicates the proenzyme MASP-3 detected with anti-mouse MASP-3 L chain Ab. Recombinant mouse MASP-1 (rMASP-1) and mouse recombinant MASP-3 (rMASP-3) were included as controls for Ab speciﬁcity. (B) Plasma MASP-3. (C) Serum MASP-3 in a complex with MBL. Each SDS-PAGE was performed under reducing conditions. All pictures are representative of three independent experiments. The Journal of Immunology 5

In conclusion, we generated mice that were monospeciﬁcally deﬁcient for MASP-1 or MASP-3 and demonstrated that MASP-3 is essentially required for the physiological activation of FD. Therefore, our study illustrates that MASP-3 plays a pivotal role in the AP via the activation of FD (Fig. 4). In addition, we demonstrated that MASP-3 circulated in an active form. However, it remains unknown how MASP-3 is physiologically activated in the circulation. Further investi- gations to elucidate the activation mechanism of MASP-3 and its role in the activation of the AP in humans are needed to complete the overall picture of the complement system.

Disclosures The authors have no ﬁnancial conﬂicts of interest.

References

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Mice deficient in ficolin, CP (i.e., C1r and C1s) and LP (i.e., MASP-1 and MASP-2) a lectin complement pathway recognition molecule, are susceptible to Streptococcus circulate as proenzymes or zymogens. The CP and LP serine pneumoniae infection. J. Immunol. 189: 5860–5866. 9. Degn, S. E., A. G. Hansen, R. Steffensen, C. Jacobsen, J. C. Jensenius, and S. Thiel. proteases are activated when PRMs in a complex with them 2009. MAp44, a human protein associated with pattern recognition molecules of bind to the ligand. In addition, the C1-inhibitor, which ex- the complement system and regulating the lectin pathway of complement activation. poses the reactive site loop mimicking the substrate specific- J. Immunol. 183: 7371–7378. 10. Pare´j, K., A. Kocsis, C. Enyingi, R. Dani, G. Oroszlań, L. Beinrohr, J. Dobo´, ity of the targeting serine proteases, regulates activation of P. Za´vodszky, G. Pa´l, and P. Ga´l. 2018. Cutting edge: a new player in the alter- the serine proteases of the CP and LP. The C1 inhibitor also native complement pathway, MASP-1 is essential for LPS-induced, but not for zymosan-induced, alternative pathway activation. J. Immunol. 200: 2247–2252. regulates the activation of the AP by interacting with C3b to 11. Xu, Y., M. Ma, G. C. Ippolito, H. W. Schroeder, Jr., M. C. Carroll, and inhibit binding of FB to C3b (18). However, neither the C1 J. E. Volanakis. 2001. Complement activation in factor D-deficient mice. Proc. Natl. Acad. Sci. USA 98: 14577–14582. inhibitor nor any other complement regulatory protein reg- 12. Iwaki, D., K. Kanno, M. Takahashi, Y. Endo, M. Matsushita, and T. Fujita. 2011. ulates or interacts with MASP-3 (19) and FD (18), thus The role of mannose-binding lectin-associated serine protease-3 in activation of the allowing them to be activated in the circulation. From these alternative complement pathway. J. Immunol. 187: 3751–3758. 13. Oroszlań, G., E. Kortvely, D. Szakaćs, A. Kocsis, S. Dammeier, A. Zeck, findings, we hypothesize that MASP-3 and FD continue to M. Ueffing, P. Za´vodszky, G. Pa´l, P. Ga´l, and J. Dobo´. 2016. MASP-1 and MASP- be active forms as they play an important role in biological ho- 2 do not activate pro-factor D in resting human blood, whereas MASP-3 is a po- tential activator: kinetic analysis involving specific MASP-1 and MASP-2 inhibitors. meostasis beyond the complement system. Indeed, we observed J. Immunol. 196: 857–865. significantly reduced body weight in the MASP-3–deficient mice 14. Oroszlań, G., R. Dani, A. Szila´gyi, P. Za´vodszky, S. Thiel, P. Ga´l, and J. Dobo´. 2017. Extensive basal level activation of complement mannose-binding lectin- compared with the WT littermates, whereas there was no signifi- associated serine protease-3: kinetic modeling of lectin pathway activation pro- cant difference in body weight between the WT and MASP-1– vides possible mechanism. Front. Immunol. 8: 1821. 15. Endo, Y., N. Nakazawa, D. Iwaki, M. Takahashi, M. Matsushita, and T. Fujita. deficient mice (Supplemental Fig. 4). In addition, gene mutations 2010. Interactions of ficolin and mannose-binding lectin with fibrinogen/fibrin in an MASP-3–specific exon, but not in MASP-1–specific exons, augment the lectin complement pathway. J. Innate Immun. 2: 33–42. are reported in patients with 3MC syndrome, which is charac- 16. Banda, N. K., M. Takahashi, B. Levitt, M. Glogowska, J. Nicholas, K. Takahashi, G. L. Stahl, T. Fujita, W. P. Arend, and V. M. Holers. 2010. Essential role of terized by unusual facial features, developmental delay, intellectual complement mannose-binding lectin-associated serine proteases-1/3 in the mu- disability, hearing loss, and slow growth after birth (20). In con- rine collagen antibody-induced model of inflammatory arthritis. J. Immunol. 185: 5598–5606. trast, FD, also known as adipsin, is mainly synthesized in adipo- 17. Kozarcanin, H., C. Lood, L. Munthe-Fog, K. Sandholm, O. A. Hamad, cytes. It was recently reported that FD has a beneficial role in A. A. Bengtsson, M. O. Skjoedt, M. Huber-Lang, P. Garred, K. N. Ekdahl, and b B. Nilsson. 2016. The lectin complement pathway serine proteases (MASPs) rep- maintaining pancreatic -cell function and may prevent the de- resent a possible crossroad between the coagulation and complement systems in velopment of type 2 diabetes (21). thromboinflammation. J. Thromb. Haemost. 14: 531–545. 6 CUTTING EDGE: ROLE OF MASP-3 IN COMPLEMENT SYSTEM

18.Jiang,H.,E.Wagner,H.Zhang,andM.M.Frank.2001.Complement1in- 20. Sirmaci, A., T. Walsh, H. Akay, M. Spiliopoulos, Y. B. Sakalar, hibitor is a regulator of the alternative complement pathway. J. Exp. Med. 194: A. Hasanefendio˘glu-Bayrak,D. Duman, A. Farooq, M. C. King, and M. Tekin. 1609–1616. 2010. MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory 19. Zundel, S., S. Cseh, M. Lacroix, M. R. Dahl, M. Matsushita, J. P. Andrieu, ﬁndings of Carnevale, Malpuech, OSA, and Michels syndromes. Am. J. Hum. W. J. Schwaeble, J. C. Jensenius, T. Fujita, G. J. Arlaud, and N. M. Thielens. 2004. Genet. 87: 679–686. Characterization of recombinant mannan-binding lectin-associated serine protease 21. Lo, J. C., S. Ljubicic, B. Leibiger, M. Kern, I. B. Leibiger, T. Moede, M. E. Kelly, (MASP)-3 suggests an activation mechanism different from that of MASP-1 and D. Chatterjee Bhowmick, I. Murano, P. Cohen, et al. 2014. Adipsin is an adipokine MASP-2. J. Immunol. 172: 4342–4350. that improves b cell function in diabetes. Cell 158: 41–53. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 A Masp1-/- Masp3-/- Intron 12 : CTTCTAACAGAATGATAAGCTGGTAACCCCAATGGGTAAG Intron 11 : GGATAGTCTGGCTAAATATGGTCCAGTCCAAAAGCGACTT |||||||||||||||||||| | | |||||||||||||||||||||| ||| | | Masp1-/- : CTTCTAACAGAATGATAAGCGAGGCTGAGAGGAAATGGGG Masp3-/- : GGATAGTCTGGCTAAATATGGTGCAGGAGACACTGACAGA | || | | || |||||||||||||||||||||||| ||| | | ||||||||||||||||||| Intron 18 : TACCAAATGGTAAGAGAAGCGAGGCTGAGAGGAAATGGGG Intron 12 : GGAACAGGCATTAGTCGAAGATGCAGGAGACACTGACAGA

B *** *** C ** 2.4 2.5 3 1.0 0.5 ** *** *** *** 2.0 0.8 0.4 1.8 2 1.5 0.6 0.3 1.2 1 mRNA (RQ) 1 mRNA (RQ) 3 mRNA 1 protein (OD) 3 protein (OD) - - 1.0 0.4 0.2 1 - - 0.6 0.5 0.2 0.1 MAp44 mRNA (RQ) MASP MASP MASP MASP

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20 0.3 0.6 10 0.5 Serum C4 (ng/ml) Serum C3 (µg/ml) 0.0 0.0 0 0 0.0 Serum ficolin Serum MBL Serum MBL WT WT WT WT WT

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Supplemental Fig. 1. Confirmation of selective deficiency for MASP-1 or MASP-3. (A) DNA sequencing analysis of the intronic recombination junctions in the MASP-1-deficient and MASP-3-deficient mice. Nucleotide sequences of the intron 12–18 junction in the MASP-1-deficient mice and the intron 11–12 junction in the MASP-3-deficient mice (middle) were aligned with mouse germline sequences (upper and lower; accession number AC163612.3). Vertical bars indicate that bases are identical between germline sequences and the query sequence. (B) Relative mRNA levels of MASP-1, MASP-3 and MAp44 were analyzed by real-time RT-PCR using mouse liver total RNAs (n = 5-6 mice per group). Values for mRNA levels are expressed as relative quantity (RQ) against the 2-ΔΔCt values in the WT mice. (C) Serum levels of MASP-1 and MASP-3 proteins analyzed by ELISA. Protein levels were expressed as OD at 450 nm analyzed in the same microplate for each (n = 3 mice per group). (D) Serum levels of C3, C4, MBL-A, MBL-C and ficolin-A proteins analyzed by ELISA. Protein level for ficolin-A was expressed as OD at 450 nm analyzed in the same microplate for each analyte (n = 3-10 mice per group). Data are represented as means ± SEM. Asterisks represent statistical differences against the values for the WT mice at *P < 0.05, **P < 0.01, or ***P < 0.001 (Dunnett’s multiple comparisons). A WT MASP-1-def MASP-3-def MASP-1/3-def

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Supplemental Fig. 2. Roles of MASP-3 in C3 deposition activity on zymosan (A) and activation of FD (B). (A) Kinetic analysis of C3 deposition activity in mouse serum on zymosan particles by flow cytometry. Histograms represent results on C3 deposition on zymosan incubated with (filled with gray) or without (open histogram) mouse sera (n = 3 mice per group). (B) The activation state of circulating FD analyzed by ELISA (n = 3 mice per group). Data are represented as means ± SEM. Asterisks represent statistical differences against the values for the WT mice at *P < 0.05, **P < 0.01, or ***P < 0.001 (Dunnett’s multiple comparisons). A B Sample 1 kDa kDa 1 MRFLSFWRLL LYHALCLALP EVSAHTVELN EMFGQIQSPG YPDSYPSDSE Proenzyme 51 VTWNITVPEG FRIKLYFMHF NLESSYLCEY DYVKVETEDQ VLATFCGRET 100 100 Sample 1 MASP-3 101 TDTEQTPGQE VVLSPGTFMS VTFRSDFSNE ERFTGFDAHY MAVDVDECKE 75 75 151 REDEELSCDH YCHNYIGGYY CSCRFGYILH TDNRTCRVEC SGNLFTQRTG Sample 2 201 TITSPDYPNP YPKSSECSYT IDLEEGFMVS LQFEDIFDIE DHPEVPCPYD 251 YIKIKAGSKV WGPFCGEKSP EPISTQTHSV QILFRSDNSG ENRGWRLSYR 301 AAGNECPKLQ PPVYGKIEPS QAVYSFKDQV LVSCDTGYKV LKDNEVMDTF 50 50 351 QIECLKDGAW SNKIPTCKIV DCGAPAGLKH GLVTFSTRNN LTTYKSEIRY 401 SCQQPYYKML HNTTGVYTCS AHGTWTNEVL KRSLPTCLPV CGQPSRALPN Sample 3 ↓ Cleavage site 37 L-chain of 451 LVKRIIGGRN AELGLFPWQA LIVVEDTSRV PNDKWFGSGA LLSESWILTA 37 501 AHVLRSQRRD NTVIPVSKEH VTVYLGLHDV RDKSGAVNSS AARVILHPDF activated 551 NIQNYNHDIA LVQLQKPVPL GAHVMPICLP RPEPEGPAPH MLGLVAGWGI MASP-3 601 SNPNVTVDEI ILSGTRTLSD VLQYVKLPVV SHAECKASYE SRSGNYSVTE 651 NMFCAGYYEG GKDTCLGDSG GAFVIFDEMS QHWVAQGLVS WGGPEECGSK 701 QVYGVYTKVS NYVDWLWEEM NSPRAVRDLQ VER WT -def WT Sample 2 -1/3 1 MRFLSFWRLL LYHALCLALP EVSAHTVELN EMFGQIQSPG YPDSYPSDSE 51 VTWNITVPEG FRIKLYFMHF NLESSYLCEY DYVKVETEDQ VLATFCGRET MASP 101 TDTEQTPGQE VVLSPGTFMS VTFRSDFSNE ERFTGFDAHY MAVDVDECKE 151 REDEELSCDH YCHNYIGGYY CSCRFGYILH TDNRTCRVEC SGNLFTQRTG 201 TITSPDYPNP YPKSSECSYT IDLEEGFMVS LQFEDIFDIE DHPEVPCPYD 251 YIKIKAGSKV WGPFCGEKSP EPISTQTHSV QILFRSDNSG ENRGWRLSYR 301 AAGNECPKLQ PPVYGKIEPS QAVYSFKDQV LVSCDTGYKV LKDNEVMDTF 351 QIECLKDGAW SNKIPTCKIV DCGAPAGLKH GLVTFSTRNN LTTYKSEIRY 401 SCQQPYYKML HNTTGVYTCS AHGTWTNEVL KRSLPTCLPV CGQPSRALPN ↓ Cleavage site 451 LVKRIIGGRN AELGLFPWQA LIVVEDTSRV PNDKWFGSGA LLSESWILTA 501 AHVLRSQRRD NTVIPVSKEH VTVYLGLHDV RDKSGAVNSS AARVILHPDF 551 NIQNYNHDIA LVQLQKPVPL GAHVMPICLP RPEPEGPAPH MLGLVAGWGI 601 SNPNVTVDEI ILSGTRTLSD VLQYVKLPVV SHAECKASYE SRSGNYSVTE 651 NMFCAGYYEG GKDTCLGDSG GAFVIFDEMS QHWVAQGLVS WGGPEECGSK 701 QVYGVYTKVS NYVDWLWEEM NSPRAVRDLQ VER Sample 3 1 MRFLSFWRLL LYHALCLALP EVSAHTVELN EMFGQIQSPG YPDSYPSDSE 51 VTWNITVPEG FRIKLYFMHF NLESSYLCEY DYVKVETEDQ VLATFCGRET 101 TDTEQTPGQE VVLSPGTFMS VTFRSDFSNE ERFTGFDAHY MAVDVDECKE 151 REDEELSCDH YCHNYIGGYY CSCRFGYILH TDNRTCRVEC SGNLFTQRTG 201 TITSPDYPNP YPKSSECSYT IDLEEGFMVS LQFEDIFDIE DHPEVPCPYD 251 YIKIKAGSKV WGPFCGEKSP EPISTQTHSV QILFRSDNSG ENRGWRLSYR 301 AAGNECPKLQ PPVYGKIEPS QAVYSFKDQV LVSCDTGYKV LKDNEVMDTF 351 QIECLKDGAW SNKIPTCKIV DCGAPAGLKH GLVTFSTRNN LTTYKSEIRY 401 SCQQPYYKML HNTTGVYTCS AHGTWTNEVL KRSLPTCLPV CGQPSRALPN ↓ Cleavage site 451 LVKRIIGGRN AELGLFPWQA LIVVEDTSRV PNDKWFGSGA LLSESWILTA 501 AHVLRSQRRD NTVIPVSKEH VTVYLGLHDV RDKSGAVNSS AARVILHPDF 551 NIQNYNHDIA LVQLQKPVPL GAHVMPICLP RPEPEGPAPH MLGLVAGWGI 601 SNPNVTVDEI ILSGTRTLSD VLQYVKLPVV SHAECKASYE SRSGNYSVTE 651 NMFCAGYYEG GKDTCLGDSG GAFVIFDEMS QHWVAQGLVS WGGPEECGSK 701 QVYGVYTKVS NYVDWLWEEM NSPRAVRDLQ VER

C H-chain L-chain MASP-3 Sample 1 Sample 2 Sample 3 ↑ Cleavage site Supplemental Fig. 3. Mass spectrometric analysis of mouse MASP-3. (A) Serum MASP-3 was isolated by immunoprecipitation using anti-mouse MASP-3 L-chain antibody prepared in this study. Immunoprecipitated MASP-3 was subjected to SDS-PAGE followed by Western blotting using anti-mouse MASP-3 L-chain antibody (left panel) or Coomassie brilliant blue (CBB)-staining (right panel). Protein bands correspond to proenzyme MASP-3 (designated as “Sample 1”), an activated MASP-3 H-chain (designated as “Sample 2”), and an activated MASP-3 L-chain (designated as “Sample 3”), all of which were estimated based on the results on Western blotting (left panel). The bands were then excised from the CBB-stained gels. (B), (C) Comparisons between amino acid sequences of full-length mouse MASP-3 obtained from the database and those of the digested fragments obtained by mass spectrometry. The excised proteins were subjected to in-gel digestion with trypsin, followed by analysis using HPLC-MS/MS (HPLC, EASY-nLC 1000; MS, Orbitrap Elite; Thermo Fisher Scientific). The amino acid sequences obtained by HPLC-MS/MS corresponding to Sample 1 (upper), Sample 2 (middle), and Sample 3 (lower) are filled in grey (B). Comparison of the sequences were summarized in a scheme (C). Red arrows indicate the cleavage site of MASP-3 when a proenzyme MASP-3 converts into an active form. Mass spectrometric analyses confirmed that proenzyme MASP-3 is detected in the region referred to as “Sample 1” located on approximately 110 kDa, H-chain of activated MASP-3 is detected in the region referred to as “Sample 2” located on approximately 70 kDa, and L-chain of activated MASP-3 is detected in the region referred to as “Sample 3” located on approximately 37 kDa. A Male B Female 30 Male 25 Female

25 20 20 MASP-1- 15 deficient mice 15 WT littermates WT littermates Body weight (g) Body weight (g) 10 MASP-1-def 10 MASP-1-def 6 8 10 12 14 6 8 10 12 14 Age (weeks) Age (weeks)

C Male D Female 30 Male 25 Female

25 20 MASP-3- 20 deficient mice 15 15 WT littermates WT littermates Body weight (g) Body weight (g) 10 MASP-3-def 10 MASP-3-def 6 8 10 12 14 6 8 10 12 14 Age (weeks) Age (weeks)

Supplemental Fig. 4. Body weight of mice. The body weight of the WT, MASP-1-deficient and MASP-3-deficient mice were recorded weekly from 6 to 14 weeks of age and plotted separately by gender. Data are represented as means ± SEM (n = 5–8). (A) Two-way ANOVA (type of genetic deficiency × age) revealed a significant effect of age (F(2.485,27.02) = 84.96, P < 0.0001), a non-significant effect of type of genetic deficiency (F(1,11) = 0.8029, P = 0.3894) and a non-significant interaction between factors (F(8,87) = 1.648, P = 0.1230). (B) Two-way ANOVA (type of genetic deficiency × age) revealed a significant effect of age (F(1.793,13.89) = 9.275, P = 0.0034), a non-significant effect of type of genetic deficiency (F(1,8) = 1.794, P = 0.2173) and a non-significant interaction between factors (F(8,62) = 0.1260, P = 0.9979). (C) Two-way ANOVA (type of genetic deficiency × age) revealed a significant effect of age (F(2.878,37.05) = 93.59, P < 0.0001), a significant effect of type of genetic deficiency (F(1,13) = 12.42, P = 0.0037) and a non-significant interaction between factors (F(8,103) = 1.730, P = 0.1002). (D) Two-way ANOVA (type of genetic deficiency × age) revealed a significant effect of age (F(3.587,37.22) = 22.28, P < 0.0001), a significant effect of type of genetic deficiency (F(1,11) = 17.37, P = 0.0016) and a non-significant interaction between factors (F(8,83) = 0.4709, P = 0.8734).