Endogenous Natural Complement Inhibitor Regulates Cardiac Development Simon A

Endogenous Natural Complement Inhibitor Regulates Cardiac Development Simon A. Mortensen, Louise L. Skov, Kasper Kjaer-Sorensen, Annette G. Hansen, Søren Hansen, Frederik This information is current as Dagnæs-Hansen, Jens C. Jensenius, Claus Oxvig, Steffen of September 27, 2021. Thiel and Søren E. Degn J Immunol 2017; 198:3118-3126; Prepublished online 3 March 2017;

doi: 10.4049/jimmunol.1601958 Downloaded from http://www.jimmunol.org/content/198/8/3118

Supplementary http://www.jimmunol.org/content/suppl/2017/03/03/jimmunol.160195 Material 8.DCSupplemental http://www.jimmunol.org/ References This article cites 45 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/198/8/3118.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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Endogenous Natural Complement Inhibitor Regulates Cardiac Development

Simon A. Mortensen,* Louise L. Skov,† Kasper Kjaer-Sorensen,† Annette G. Hansen,* Søren Hansen,‡ Frederik Dagnæs-Hansen,* Jens C. Jensenius,* Claus Oxvig,† Steffen Thiel,* and Søren E. Degn*,x

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease–pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue Downloaded from strategies in zebraﬁsh, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development. The Journal of Immunology, 2017, 198: 3118–3126. http://www.jimmunol.org/

eart development is a complex process involving cell in nonconsanguineous parents, supporting the existence of im- speciﬁcation and differentiation, as well as elaborate tis- portant genetic components (3). H sue morphogenesis and remodeling to generate a func- Several recent studies reported that mutations in the immune- tional organ. Congenital heart disease (CHD) is the most common related molecules mannan-binding lectin (MBL)-associated ser- cause of major congenital anomalies, representing a signiﬁcant ine protease (MASP)-3 and collectin-kidney 1 (CL-K1) underlie global health problem (1). The reported birth prevalence of CHD the etiology of the Michels, Malpuech, Mingarelli, and Carnevale

varies widely among studies worldwide. The estimate of 8 per (3MC) syndrome (4–6). 3MC syndrome is a rare congenital dis- by guest on September 27, 2021 1000 live births is generally accepted as the best approximation; order that is most commonly associated with consanguinity. It is as such, CHD accounts for nearly a third of all major congenital characterized by developmental defects including, among others, anomalies (2). Not surprisingly, CHD birth prevalence among growth retardation, craniofacial abnormalities, midline defects, children with consanguineous parents is considerably higher than and caudal appendage. Recent observations added CHD to this list (6). It seems plausible that the causative mutations in CL-K1 or MASP-3 are based in a physiologically relevant complex of the *Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; two proteins, but the exact nature and role of such a complex, as †Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; ‡Department of Cancer and Inflammation Research, University of Southern well as the relevant substrate of MASP-3, remain unclear (7). Denmark, DK-5000 Odense, Denmark; and xProgram in Cellular and Molecular Medicine, However, a recent structure of the catalytic domain of zymogen Boston Children’s Hospital, Boston, MA 02115 MASP-3 indicates that a 3MC-associated mutation perturbs the ORCIDs: 0000-0001-9013-1802 (S.A.M.); 0000-0002-8186-4630 (S.H.). structure of the catalytic site of this protease (8), suggesting that Received for publication November 17, 2016. Accepted for publication February 9, active MASP-3 is a prerequisite for normal development. CL-K1, 2017. a member of the collectin family of collagen-containing lectin This work was supported by the Lundbeck Foundation (to S.E.D. and S.T.). proteins, was proposed to serve as a guidance cue for neural crest The sequences presented in this article have been deposited in the European Nucle- cell migration (4). It is believed to be a pattern recognition mol- otide Archive (http://www.ebi.ac.uk/ena/data/view/LK939138; http://www.ebi.ac.uk/ ena/data/view/LK939139) under sequence numbers LK939138 and LK939139. ecule (PRM), but little is known about the physiological ligands Address correspondence and reprint requests to Dr. Søren E. Degn, Department of Bio- and functions of CL-K1 (9). Furthermore, the majority of CL-K1 medicine, Bartholins Alle´ 6, Building 1242, Room 565, Aarhus University, DK-8000 is found in the circulation as a heterocomplex with the related Aarhus C, Denmark. E-mail address: [email protected] molecule collectin-liver 1; this form is termed collectin-L1/K1 The online version of this article contains supplemental material. hetero-oligomer (CL-LK) (10). MASP-3 is one of three alterna- Abbreviations used in this article: CHD, congenital heart disease; CL-K1, collectin- tive splice products (MASP-1, MASP-3, and MBL-associated kidney 1; CL-LK, collectin-L1/K1 hetero-oligomer; cMO, control MO; dpf, day postfertilization; hpf, hour postfertilization; ISH, in situ hybridization; MAp, MBL- protein [MAp]44) from the human MASP1 gene (11). MASP-1 associated protein; MASP, MBL-associated serine protease; MBL, mannan-binding and MASP-3 are serine proteases, whereas MAp44 is a truncated lectin; 3MC, Michels, Malpuech, Mingarelli, and Carnevale; MO, morpholino; MRP, MASP-related protein; PBT, PBS, 0.01% Tween 20; PRM, pattern recognition product retaining only the domains required for association with molecule; rh, recombinant human; RT-qPCR, reverse transcription–quantitative PCR; PRMs. This raised the possibility that MAp44 is able to regulate 2+ rz, recombinant zebrafish; TBS/Tw/Ca , TBS, 0.05% (v/v) Tween 20, 5 mM CaCl2 the composition of PRM/MASP complexes (12). Although the (pH 7.4). exons encoding the MASP-1 protease domain in the MASP1 gene Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 have been secondarily lost in birds and fish, those of MASP-3 and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601958 The Journal of Immunology 3119

MAp44 are highly conserved evolutionarily (13, 14). MAp44 was Table I). The construct was verified by sequencing and deposited in the originally identified in carp as a novel truncated isoform of MASP European Nucleotide Archive under sequence number LK939139 (http:// named MASP-related protein (MRP) (15). www.ebi.ac.uk/ena/data/view/LK939139). In addition to the zMASP3 construct, an inactive version, zMASP3i, was made by mutating the cata- Prior studies indicated a high expression of MAp44 in the de- lytic site serine to an alanine. Mutagenesis was performed using a Quik- veloping human heart (11, 16). In adult mice, administration of Change II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa exogenous MAp44 attenuated myocardial injury and arterial Clara, CA), according to the manufacturer’s instructions. Primers P5 and thrombogenesis (17). To examine a potential function of MAp44 P6 were used. Recombinant zebrafish (rz)MASP-3i was used in competition experiments because of good expression yield and high stability. in the developing heart, we used morpholino (MO) knockdown and rescue strategies in zebrafish. The zebrafish has emerged as a Pseudophylogenetic analysis of MASP-1, MASP-3, and MAp44 powerful model system to unravel the basic genetic, molecular, The serine protease domain for MASP-1, MASP-3, and the unique C and cellular mechanisms of cardiac development and function (18, terminus for MAp44 were aligned using the muscle algorithm in the 19). By 30 h postfertilization (hpf), the transparent, rapidly de- software package SeaView 4.5.0 (23). This software was also used to veloping zebrafish embryo has a complete functioning circulatory construct an unrooted phylogenetic tree to investigate the relationship network. Because of the small size of the embryo, it can obtain among the proteins. The protein sequences were randomized five times, and the parsimony algorithm was used to assess the phylogenetic rela- sufficient oxygen by passive diffusion up until about day 3 of tionship between orthologs of the proteins. The serine protease domains development, allowing assessment of conditions severely per- for MASP-1 and MASP-3 were defined using the Pfam algorithm (24), and turbing heart function without causing lethality. Although the the C terminus for MAp44 was defined as the sequence downstream of the zebrafish has a single-loop circulatory system, cardiac development CCP1 domain. appears to closely reflect that in higher vertebrates with divided Expression and purification of recombinant and natural Downloaded from systemic and pulmonary circulatory systems. Strong similarities proteins in the function of cardiac neural crest cells were observed between zebrafish and mouse/man (20). Cardiac neural crest cells The cell line HEK293-F (Invitrogen) was used for transient expression of are a transient, migratory cell population that is exclusive to ver- rzMAp44 and rzMASP-3. HEK293-F cells were grown in FreeStyle 293 Expression medium (Life Technologies) with agitation at 37˚C and 8% tebrate embryos. They play an essential role in the remodeling CO2. Transfection was done using Lipofectamine 2000 Transfection Re- of the initially bilateral and symmetric pharyngeal artery pairs agent (Invitrogen), following the manufacturer’s instructions. After trans- http://www.jimmunol.org/ into an aortic arch and in the septation of the cardiac outflow tract fection, the cells were grown for 72 h, and the supernatant was harvested into the base of the pulmonary artery and aorta. Accordingly, by centrifugation. Recombinant proteins were purified by affinity chromatography, as done defective cardiac neural crest cell function is a common cause previously for human MAp44 (11), using recombinant human (rh)MBL of congenital heart defects (21). coupled to Sepharose beads. Supernatant harvested from cells express- ing rzMAp44 was supplemented with 5 mM CaCl2 and incubated with rhMBL-coupled beads for 2 h at 4˚C at a 1:5 ratio (beads/supernatant). Materials and Methods 2+ Zebrafish After incubation, beads were washed in TBS/Ca (10 mM Tris, 145 mM NaCl, 5 mM CaCl2 [pH 7.4]), followed by elution of the bound rzMAp44 Zebrafish of the Tubingen€ and AB strains were fed four times daily and with TBS, 10 mM EDTA, and 1 M NaCl (pH 7.4). The purity of the eluted housed at 28˚C with a 14-h light/10-h dark cycle. The Tubingen€ strain was protein was checked by SDS-PAGE, followed by silver staining; the by guest on September 27, 2021 used for cloning of zebrafish MAp44 and MASP-3, whereas the AB strain protein-containing fractions were pooled and concentrated using a Viva- was used in the remainder of the experiments. Embryos were obtained by spin 6 (10-kDa molecular mass cut-off; Sartorius) and buffer exchanged 2+ natural crosses, reared in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM into TBS/Ca using a Vivaspin 6 concentrator. Final protein concentration CaCl2,0.33mMMgSO4, 0.00001% methylene blue, 2 mM HEPES [pH 7]) was estimated by A280 (using a calculated extinction coefficient of 1.08 at 28.5˚C, and staged as described (22). Embryos were sedated in tricaine for a 1-mg/ml concentration). In a similar manner, we also expressed methanesulfonate (MS-222, 150 ng/ml; Sigma-Aldrich) when required for rzMASP-3i, rhMAp44, and rhMASP-3 and purified these on MBL-coupled phenotypic analysis and were euthanized by tricaine overdose. All experi- beads, as described (11). ments involving zebrafish embryos and larvae were carried out according to Naturally occurring CL-LK was purified from plasma by calcium- Danish legislation. sensitive immunoaffinity chromatography, as previously described (25). Cloning of mRNA encoding zebrafish MAp44 and MASP-3 Competitive binding to rhMBL or CL-LK RNAwas extracted from 6 d postfertilization (dpf) whole zebrafish embryos Microtiter plates were coated overnight at 4˚C with rhMBL (5 mg/ml) (genetic background Tubingen)€ using TRIzol Reagent (Invitrogen), fol- in coating buffer (0.1 M sodium bicarbonate [pH 9.6]). The next day, lowing the manufacturer’s instructions. To clone mRNA encoding zebra- microtiter plates were emptied and incubated for 1 h at room temperature fish MAp44, a second-generation 59/39 RACE kit (Roche) was used. cDNA in TBS, 0.05% (v/v) Tween 20 (pH 7.4). Microtiter plates were washed + 2+ poly(A) synthesis was carried out using 1 mg of total RNA and an oligo twice with TBS, 0.05% (v/v) Tween 20, 5 mM CaCl2 (pH 7.4) (TBS/Tw/Ca ) (dT) primer with an anchor sequence. Gene-specific cDNA amplification before use. A constant concentration of rhMAp44 (0.1 mg/ml) was was performed with a generic anchor primer and primer P1 specific for mixed with decreasing concentrations (starting at 20 mg/ml and 2-fold zebrafish MASP1 (Supplemental Table I). PCR was set up as follows: dilutions thereof) of rzMAp44 in binding buffer (20 mM Tris, 1 M NaCl, initial denaturation at 98˚C for 30 s, 35 cycles of 98˚C for 10 s and 72˚C 0.05% Triton X-100, 10 mM CaCl2, 0.01 mg/ml HSA [pH 7.4]) before for 90 s, and a final extension for 10 min at 72˚C. The PCR product from incubation of the mixture overnight at 4˚C in the rhMBL-coated microtiter the above reaction was used as the template for nested PCR with primers wells. The following day, microtiter wells were washed three times with P2 and P3 (Supplemental Table I). The PCR cycling conditions were as TBS/Tw/Ca2+ before incubating for 2 h at room temperature with biotin- above with the exception of the extension time, which was reduced to 45 s. labeled Ab directed against hMAp44 (clone 2D5) (26). After incubation, One amplicon, detected by agarose gel electrophoresis and purified using the microtiter wells were washed three times with TBS/Tw/Ca2+ and de- the QIAquick Gel Extraction Kit (QIAGEN), was digested with XbaI/KpnI veloped with 250 ng/ml Eu3+-labeled streptavidin (PerkinElmer) in TBS, and ligated into a pcDNA3.1/myc-His(-) A vector (Invitrogen) using a 0.05% (v/v) Tween 20, 25 mM EDTA (pH 7.4) for 1 h. After incubation, Quick Ligation Kit (NEB). Prior to ligation, the vector was digested with microtiter wells were washed three times with TBS/Tw/Ca2+ and added XbaI/KpnI and dephosphorylated with calf intestinal alkaline phosphatase enhancement solution (PerkinElmer), and the amount of Eu3+ in the (NEB). The final construct was verified by sequencing and deposited in the microtiter wells was read by time-resolved fluorometry. European Nucleotide Archive under sequence number LK939138 (http:// Similar set-ups were used for assaying the binding and competitive- www.ebi.ac.uk/ena/data/view/LK939138). The sequence is seen aligned binding activity of MASP-3 and MAp44 to rhMBL and purified human with the human MAp44 sequence in Supplemental Fig. 1C. CL-LK. Detection of zebrafish MASP-3 was performed using an in-house– The cDNA used to clone zMAp44 also was used to clone zMASP3. The generated biotinylated rabbit polyclonal anti-zebrafish MASP-3 Ab raised approach was as described for zMAp44, with the exception that gene- against the peptide NYLQWLHTHMDAER, whereas human MAp44 and specific primers P2 and P4 were used to amplify zMASP3 (Supplemental MASP-3 were detected with biotinylated mAb 38.12.3 and mAb 2D5, 3120 COMPLEMENT INHIBITOR MAp44 REGULATES CARDIAC DEVELOPMENT respectively (26). To evaluate the binding of human MAp44, human MAp44 ISH probe MASP-3, and zebrafish MASP-3 to CL-LK, 50, 3.3, or 0 mg/ml of each 9 9 protein was added to CL-LK–coated microtiter wells. For each developing The PCR product (generated using the 5 /3 RACE kit, see above) that was Ab, readings were normalized to the background (0 mg/ml). used to clone zebrafish MAp44 was also used to generate a MAp44 ISH probe using primers P11 and P12. The PCR was set up as follows: initial Mouse hearts denaturation at 98˚C for 30 s, 35 cycles of 98˚C for 10 s and 72˚C for 15 s, and a final extension for 5 min at 72˚C. The amplicon was purified from an C57BL/6J BomTac mice were obtained from Taconic Europe (Ry, Denmark). agarose gel using a QIAquick Gel Extraction Kit (QIAGEN) and ligated The mice were 9–12 wk old with a bodyweight of ∼19–22 g for females and into the pSC-B-amp/kan vector (Stratagene), following the manufacturer’s 25–29 g for males when used for mating. Female mice were housed in open instructions. The construct was verified by sequencing. cages in groups of five or six (type III plastic cages; Tecniplast, Buguggiate, To obtain the antisense strand, the plasmid was digested with XbaI Italy) until mating. Males were housed singly (type II plastic cages; Tecni- and PciI (T3 promoter antisense), whereas the sense strand was ob- plast), except for when they were joined by females to set up matings. The tained by digesting with NcoI and HindIII (T7 promoter sense). In both animal room had a 12-h light/12-h dark schedule, with a temperature of 21 6 cases, the product was purified from an agarose gel using a NucleoSpin 2˚C and 55 6 10% relative humidity. The animals were fed standard chow Gel and PCR Clean-up Kit (MACHEREY-NAGEL), following the (Altromin 1324; Altromin Spezialfutter, Lage, Germany) and water ad manufacturer’s instructions. Transcription was conducted with T3 libitum throughout the experiment. Mice were given nesting material, polymerase (Fermentas) or T7 polymerase (Promega) and DIG RNA shredded paper strips, and wooden squares as environmental enrichment. labeling mix (Roche), according to manufacturer’s protocol. The RNA Bedding was aspen wood chips from Tapvei, Finland. Animal rooms were probe was purified using SigmaSpin Post-Reaction Clean-Up columns monitored two times a year for common murine pathogens. Timed preg- (Sigma). nancies were set up; at the indicated time points, pregnant females were euthanized by cervical dislocation, and their fetuses were dissected from the crip2 ISH probe uterus and, as for newborn pups, euthanized by decapitation. Hearts were Downloaded from surgically extracted using a dissection microscope, placed in TRI Reagent The coding sequence of crip2 (GenBank accession no. BC044391.1, solution (Ambion), and frozen at 280˚C until further processing. All ex- https://www.ncbi.nlm.nih.gov/nuccore/BC044391.1) was synthesized by GenScript and cloned into the pUC59 vector using EcoRI and XbaI. The periments involving mice and housing of the mice were carried out 9 according to Danish legislation and Directive 2010/63/EU on the protection following sequence was placed upstream of the start-codon (5 -AAT- of animals used for scientific purposes. TAACCCTCACTAAAGGGAACAAAAGCTGGGTACCGGGCCCCC- CCTCGAGGTCGACGGTATCGATGATATCCACTGTGGCCCTT-39) RNA extraction and cDNA synthesis including a T3 promoter and additional restriction sites. Another sequence 9

(5 -AAGGGCCACATTGGTCGCTGCAGCCCGGGGGATCCACTA- http://www.jimmunol.org/ Total RNA was extracted from embryos, adult zebrafish, and mouse hearts, GTCCCTATAGTGAGTCGTATTAC-39) containing additional restric- using TRI Reagent solution (Ambion) combined with the PureLink RNA tion sites and a T7 promoter was placed downstream of the stop codon. To Micro Scale Kit (Invitrogen), according to the manufacturer’s instructions, obtain the sense strand, the plasmid was digested with NdeI and BamHI and RNA concentration was measured by spectrometry. For cDNA syn- (T3 promoter sense), and to obtain the antisense it was digested with XhoI thesis, 1 mg of total RNA was used with an iScript cDNA Synthesis Kit and HindIII (T7 promoter antisense), followed by purification from an (Bio-Rad). agarose gel using a NucleoSpin Gel and PCR Clean-up Kit. As above, transcription was conducted with T3 polymerase or T7 polymerase and Reverse transcription–quantitative PCR DIG RNA Labeling Mix. The RNA probe was purified using SigmaSpin Post-Reaction Clean-Up Columns. The reverse transcription–quantitative PCR (RT-qPCR) reaction was per-

formed using a SensiFAST SYBR Lo-ROX Kit (Bioline) on a Stratagene cmlc2 (myl7) ISH probe by guest on September 27, 2021 Mx3005P with MxPro QPCR Software (both from Agilent Technologies). Cycling conditions were as follows: 2 min at 95˚C, followed by 40 cycles of A cmlc2 (myl7) probe for whole-mount ISH was generated. A 500-bp 5 s at 95˚C, 10 s at 60˚C, and 5 s at 72˚C. MAp44 transcript levels in larvae fragment of the zebrafish gene encoding cmlc2 was chosen. This region and adult tissue were normalized to EEF1a (ENSDART00000023156), was amplified in a nested PCR using primers (cmlc2_outer_fwd: 59- and primers were designed using GeneFisher2 (27). Primer sequences TGTGCAGTTATCAGGGCTCCTGT-39, cmlc2_outer_rev: 59-GGGCCTTA- for MAp44 P7/P8 and EEF1a P9/P10 can be found in Supplemental AACCAAATGTCTAGTTA-39, cmlc2_inner_fwd: 59-TAGGAGGCTCT- Table I. GGGTGTCCATGT-39, and cmlc2_inner_rev: 59-GGGCAGCAGTTA- RT-qPCR analysis of MAp44 expression in mouse hearts was done CAGACAGAATAA-39). The final product was purified, and T-overhangs using P13/P14 and a TaqMan probe P15 for MAp44. Primers and Taq- were generated to allow ligation into the pGEM-T vector (Promega), as Man probe sequences can be found in Supplemental Table I. Gusb was described in the manufacturer’s protocol. The template was linearized by used as reference gene (Mm01197698_m1; Thermo Fisher Scientific). A NcoI and SspI to generate fragments with only one promoter (T7). Tran- SensiFAST Probe Lo-ROX kit (Bioline) was used with the following scription was conducted with the T7 polymerase (Promega) and DIG RNA cycling conditions: 3 min at 95˚C, followed by 45 cycles of 10 s at 95˚C Labeling Mix (Roche), according to the manufacturer’s protocol. RNA and 30 s at 60˚C. probes were purified using SigmaSpin Post-Reaction Clean-Up Columns (Sigma). MO injection Quantification of crip2 imaging data MOs (Gene Tools) and in vitro–transcribed mRNA for injection were di- luted in nuclease-free water (pH 7) and a phenol red solution. Five All images were opened in Fiji (30) and stacked. Next, a montage was made nanoliters were injected into the yolk sac at the one- to four-cell stage. A of the images. A rectangular box (equal size for all images) was selected sequence-specific MO was designed to knockdown zebrafish MAp44 by around the hearts. The montage was changed to 8-bit, and brightness/ targeting the acceptor splice site at the boundary between intron 8 and contrast was set to 0–155. The mean gray value was measured for all exon 9 (MAp44 MO: 59-TCCTTCTCTGGTGGCAGCAAAGAGA-39). hearts. To ensure that the phenotypes observed were caused by the MAp44 Heart rate recording MO and were not due to upregulation of the p53 apoptosis pathway, a p53-specific MO (p53 MO: 59-GCGCCATTGCTTTGCAAGAATTG-39) Embryos were immobilized in 3% methylcellulose on a concave glass slide was coinjected with the MAp44 MO. A standard control MO (cMO) or in 1% low-melt agarose (SeaPlaque). Recordings of heart activity were (59-CCTCTTACCTCAGTTACAATTTATA-39) was used as a negative acquired using a Hamamatsu Oraca Flash 4.0 camera mounted on a Zeiss control. Every batch of MAp44 MO was titrated down in 0.5-ng decre- microscope. Videos were recorded for 20 (or 4) s at a frame rate of 100 per ments to the minimal dose yielding an appreciable phenotype, which was second. To determine heart rates, an algorithm was written in MATLAB consistently found to be 3 ng. cMO and p53 MO were injected using the (MathWorks, Natick, MA), as described previously (31). The supplemental same dose as for the MAp44 MO. videos were acquired using a Zeiss Axio Observer.Z1 microscope equipped with an AxioCam HRm camera (frame rate of five per second). In situ hybridization Alcian blue staining In situ hybridization (ISH) with the various probes described below was carried out as described previously in detail (28). The polymer dextran The cartilage of 5 dpf old larvae was stained with Alcian blue using a sulfate was added to the hybridization step, as described previously (29). protocol modified from Neuhauss et al. (32). Briefly, embryos were The Journal of Immunology 3121 euthanized by a tricaine methanesulfonate (MS-222) overdose and fixed in domain structure conserved between zebrafish and man (Fig. 1A, 4% paraformaldehyde overnight at 4˚C. The next day, the larvae were Supplemental Fig. 1A–C). To evaluate the functional conservation washed three times with PBS, 0.01% Tween 20 (PBT) and bleached in of MAp44 in zebrafish, we expressed rzMAp44 and assessed its PBS containing 3% H2O2 and 1% KOH at room temperature to remove pigmentation. After decoloration, larvae were washed three times in PBT ability to bind human MBL (a CL-K1–related collectin) as a and stained with Alcian blue solution (0.02% Alcian blue, 80% EtOH, and model PRM (7). Notably, the interaction between MASPs and the 20% acetic acid) overnight at 4˚C. The following day, larvae were various PRMs has been shown to be very similar as a result of the destained with acidic ethanol (70% EtOH and 5% HCl in PBT) and highly conserved motifs in the binding region of both components thereafter rehydrated by a series of 30-min room temperature incubations in 75% EtOH/25% PBT, 50% EtOH/50% PBT, 25% EtOH/75% PBT, and (33). We found that zebrafish MAp44 expressed well in HEK293 finally 100% PBT. Next, the larvae were washed three times in PBS and cells and displayed a migration pattern on SDS-PAGE that was cleared overnight in PBS containing 0.05% trypsin at 4˚C. Larvae were comparable to that of rhMAp44 (Fig. 1B). As predicted based on then washed three times in PBS and fixed in PBS containing 4% para- the high degree of sequence similarity (Supplemental Fig. 1C), formaldehyde for 2 h at room temperature. Larvae were finally washed zebrafish MAp44 had the ability to form complexes with human three times in PBT and stored in 100% glycerol until further examination. MBL; this was shown in a competition assay in which rzMAp44 In vitro–transcribed MAp44 mRNA efficiently outcompeted rhMAp44 for binding to rhMBL (Fig. For rescue experiments, capped zebrafish MAp44 mRNA was synthesized 1C). We concluded that zebrafish MAp44 was functionally con- using an mMESSAGE mMACHINE T7 ULTRA Kit (Ambion). Template served in its ability to interact with PRMs. We next examined (plasmid encoding zebrafish MAp44) was linearized with NdeI and HindIIII the expression profile of mRNA encoding zebrafish MAp44 (both from NEB) and gel purified prior to in vitro transcription. The throughout development. As shown in Fig. 1D, MAp44 transcript transcribed and capped mRNA was purified using an RNeasy MinElute was expressed during early development and peaked at 48 hpf, Downloaded from Cleanup Kit (QIAGEN). RNA yield was measured by spectrophotometry, and the quality of the RNA was ascertained by gel electrophoresis. MAp44 after which it decreased. MASP3 was found to be broadly ex- mRNA was titrated down to the minimal effective dose for rescue (250 pg, pressed in rat and chick embryos by ISH (13). For comparison complete rescue with ∼90% penetrance) when coinjected with 3 ng of with MAp44, we also measured MASP3 transcript during zebrafish MAp44 MO. development. Although present during this time window, the ex- Data representation and statistics pression level was much lower than that of MAp44 (Supplemental

Fig. 1D). Prior studies, primarily based on the analysis of fetal http://www.jimmunol.org/ Data were graphed in GraphPad Prism 6.0. Statistical analyses were per- tissue, reported high expression of MAp44 in the human heart (11, formed using Prism, as indicated throughout the text and figure legends. 16). Focused analysis revealed particularly high expression of MAp44 in the developing heart of zebrafish compared with adult Results heart tissue. At both time points, MAp44 expression was .10-fold To investigate the potential role of MAp44 in cardiac development, higher than MASP3 expression in the heart (Fig. 1E). We extended we used the zebrafish model, which permits visualization of the these findings by also examining the expression profiles of MAp44 developing heart. Based on sequence homology and domain pre- and MASP3 in heart tissue in developing mouse embryos and diction, MAp44 is well conserved throughout evolution, with neonates. Again, MAp44 transcript was highly expressed in heart by guest on September 27, 2021

FIGURE 1. Functional conservation of human and zebrafish MAp44. (A) An outline of the zebrafish masp1 gene, with exons indicated by colored boxes and introns represented as black lines (not drawn to scale). The domains of resulting protein products are colored to correspond to the exons encoding the domains. (B) Purified rhMAp44 (lane 1) and zebrafish MAp44 (lane 2) analyzed by SDS-PAGE and silver staining. The migration positions of molecular mass markers are indicated on the left. (C) Competition between rhMAp44 and zebrafish MAp44 for binding to MBL. rhMAp44 was kept at a constant concentration and mixed with increasing concentrations of rzMAp44. The amount of human MAp44 bound to MBL-coated microtiter wells was measured with a human MAp44–specific Ab. (D) Measurement of zebrafish MAp44 mRNA during embryonic development (from 1 to 144 hpf) using RT-qPCR. Fifty embryos were pooled and measured in triplicate, bars represent mean 6 SD. (E) Measurement of zMAp44 transcript and zebrafish MASP-3 transcript in 48 hpf and adult hearts (mean 6 SD of triplicate measurements of eight pooled 48 hpf hearts and four pooled adult hearts). (F) Measurement of MAp44 and MASP3 mRNA in the mouse heart during embryonic and neonatal development, from embryonic day 15.5 to 28 d postpartum using RT-qPCR. Two hearts were used for each time point and were measured in triplicate; bars represent mean 6 SD. SP, serine protease. 3122 COMPLEMENT INHIBITOR MAp44 REGULATES CARDIAC DEVELOPMENT tissue during development, whereas MASP3 transcript was present 2Dk, 2Do, 2Ds) (34). Importantly, the MAp44-knockdown phe- but at much lower levels (Fig. 1F). notype could be completely reversed by coadministration of To directly address the physiological role of MAp44 expression MAp44 mRNA (in a dose-dependent manner, with a penetrance during development, we used MO-based knockdown to target the ∼ 90% at 250 pg), circumventing the MO splice block and unique intron–exon junction of the MAp44-encoding transcript thereby demonstrating a specific role for MAp44 in the observed (Fig. 2A). We verified that the MO was able to suppress expres- phenotype(Fig.2Dd,2Dh,2Dl,2Dp,2Dt). sion of MAp44, as tested after 48 and 72 hpf (Fig. 2B), and that To further investigate the observations of brain and cardiac it did not influence expression of the other splice product from edema, we conducted detailed studies of heart morphology and the gene, which encodes MASP-3 (Fig. 2C). Although knockdown function in MAp44-knockdown larvae compared with larvae in- of MAp44 at 48 hpf was nearly complete, it was incomplete at jected with cMO. This revealed a dramatic cardiac malformation 72 hpf, likely as a consequence of the progressive loss and dilution at 48 hpf following MAp44 knockdown, as visualized by bright- of the MO and the cumulative accumulation of MAp44 transcript. field microscopy, as well as by the use of cmlc2 as a heart-specific However, based on the developmental expression profile (Fig. 1E), antisense probe visualizing the looping of the heart (Fig. 3A, 3B). we anticipated 72 hpf to be at the back end of the physiological We found the knockdown to have severe functional conse- process involving MAp44 function. In agreement, when assessing quences, including decreased heart rate and evidence of heart the in vivo response to MAp44 knockdown, we saw a striking insufficiency, such as pericardial edema. To quantify the heart phenotype, with hallmarks reminiscent of human 3MC syndrome rate defect, we used video-rate imaging and developed an algorithm (Fig. 2D). This included growth retardation, body curvature, for measuring the changing pixel intensity at the boundary of the pigmentation defects, and craniofacial abnormalities (Fig. 2Db, heart, allowing precise measurement of the heart rate (Fig. 3C, Downloaded from 2Df, 2Dj, 2Dn, 2Dr, Supplemental Fig. 2). Interestingly, we also Supplemental Videos 1, 2). This revealed a profound decrease in saw a loss of pectoral fin development. The embryos displayed the heart rate of MAp44-specific MO–injected larvae compared evidence of brain and cardiac edema, indicating a possible defect with larvae injected with cMO (70.2 and 119.4 beats per minute, in heart function. No abnormalities were observed upon injection respectively (Fig. 3D). The mean diastolic interval was 0.32 6 of a standard cMO (Fig. 2Da, 2De, 2Di, 2Dm, 2Dq), and coin- 0.05 s for control and 0.39 6 0.1 s for MAp44 MO (Fig. 3E). The

jection of a p53-blocking MO with the MAp44-targeting MO did mean systolic interval was 0.17 6 0.03 s for control and 0.25 6 http://www.jimmunol.org/ not reverse the phenotype, indicating that the observed phenotype 0.07 s for MAp44 MO (Fig. 3F). These changes in the diastolic was not simply a result of nonspeciﬁc MO toxicity (Fig. 2Dc, 2Dg, and systolic intervals in the MAp44 MO group were proportional, by guest on September 27, 2021

FIGURE 2. MO-mediated MAp44 knockdown results in severe developmental defects during embryo development. (A) Illustration of the positioning of the MAp44-specific MO (MAp44 MO) used to knock down MAp44 expression. Exons are represented as colored boxes, and introns are represented as black lines (not drawn to scale). The exons are colored corresponding to the domains that they encode as follows: CUB1 and CUB2 (blue), EGF (orange), CCP1 and CCP2 (gray), MASP-3 serine protease (SP) domain (red). The positions of the primers used to verify knockdown of MAp44 by RT-qPCR are also shown. (B) RT-qPCR measurement of the MAp44 transcript in zebrafish injected with cMO or the MAp44 MO at 48 and 72 hpf. Bars represent mean 6 SD of a pool of 30 embryos measured in triplicate. Data normalized to cMO and statistical significance were determined by two-way ANOVA and the Sidak posttest. (C) RT-qPCR measurement of the zMASP3 transcript in zebrafish injected with a cMO or the MAp44 MO at 48 hpf. Bars represent mean 6 SD for 30 embryos measured in triplicate for each experiment. Data normalized to cMO and statistical significance were determined using an unpaired t test with the Welch correction (two-tailed p value = 0.1533). (D) Images (representative of $30 embryos for each condition) of larvae at 22, 48, and 72 hpf injected with cMO (a, e, i, m, q), MAp44 MO (b, f, j, n, r), MAp44 MO coinjected with p53 MO (c, g, k, o, s), or MAp44 MO coinjected with 250 pg MAp44 mRNA (d, h, l, p, t). Arrows in (j) point to head and heart edema, and arrow in (r) points to a fin bud from which the pectoral fin normally develops. ****p , 0.0001 (multiplicity-adjusted). n.s., not significant. The Journal of Immunology 3123 as revealed when normalizing to the heart period (Supplemental used as a model PRM). Indeed, zebrafish MAp44, like human Fig. 3A). The phenotype was completely rescued by coadminis- MAp44, competed with MASP-3 for binding to MBL (Fig. 5A). tration of MAp44 mRNA, again demonstrating MAp44 causality Next, we examined whether zebrafish MASP-3 was able to bind in the observed phenotype (Fig. 3D). human CL-LK, as a surrogate for the binding ability of zebrafish As mentioned earlier, CL-K1 and MASP-3 were suggested to MAp44, because no anti–zebrafish MAp44 Ab is available. We influence migration and function of neural crest cells (4). Neural expressed and purified rzMASP-3 (Supplemental Fig. 3B) and crest cells are involved in the genesis of craniofacial cartilage verified the reactivity of our anti–zebrafish MASP-3 Ab (Supplemental structures, a subset of pigment cells, and cardiac development Fig. 3C). Zebrafish MASP-3, like human MAp44 and human (35). Whole-mount ISH revealed notable expression of MAp44 in MASP-3, displayed titratable binding to immobilized human the midbrain and hindbrain, and we observed craniofacial abnor- CL-LK (Fig. 5B). Finally, we examined whether zebrafish MAp44 malities following MAp44 knockdown, as visualized by Alcian was able to compete with zebrafishMASP-3intheinteraction blue staining (Supplemental Fig. 2). To investigate a potential role withCL-LK.AscanbeseeninFig.5C,thiswasindeedthe for neural crest cells in the observed MAp44-knockdown pheno- case, leading us to propose a mechanism of function whereby type, we conducted ISH with a probe for cysteine-rich protein 2 MAp44 competitively regulates the level of active CL-LK/MASP-3 (crip2), a marker of cardiac neural crest cells during early em- complexes. bryogenesis (36), on 27 hpf cMO- or MAp44 MO–injected embryos (Fig. 4). Knockdown of MAp44 caused significantly Discussion stronger signal, indicating aberrant cardiac neural crest cell Knockdown of MAp44 nearly phenocopies the phenotype observed behavior. by Rooryck et al. (4) upon “masp1 knockdown,” although a heart Downloaded from As shown above, MAp44 appears to be involved in the biology of phenotype was not noted in their work. They observed pigment neural crest cells, and MASP-3 deficiency was reported to influence and craniofacial cartilage defects, including reduced mandibular neural crest cell migration (4). In addition, MAp44 was demon- length resulting from shortened Meckel’s and palatoquadrate strated to compete with MASPs for binding to PRMs in vitro (11, cartilages. We also observed these craniofacial defects following 12) and in vivo (17). Therefore, we hypothesized that MAp44 MAp44 knockdown and additionally noted the heart phenotype,

functions in vivo by regulating the amount of MASP-3 associated loss of the pectoral fin, and brain edema. As mentioned previously, http://www.jimmunol.org/ with PRMs. Accordingly, we first examined whether zebrafish the zebrafish masp1 gene does not encode MASP-1, only MASP-3 MAp44 would compete with MASP-3 for binding to MBL (again and MAp44. The MO used by Rooryck et al. (4) targeted both of by guest on September 27, 2021

FIGURE 3. MAp44 knockdown induces severe cardiac defects. (A) Representative bright field microscopy photograph of a heart at 48 hpf from an embryo injected with cMO or MAp44 MO. (B) Whole-mount ISH of cMO- and MAp44 MO–injected larvae at 48 hpf using a heart-specific antisense probe (cmlc2) visualizing the looping of the heart. (C) Examples of changing pixel intensity and M-modes for 48-hpf embryos injected with cMO or MAp44 MO. The changing pixel intensity displays peaks at the onset of diastole and systole (*). The M-mode shows the embryonic ventricle movement. (D) Heart rate measurements from high-speed video recording of embryos injected with cMO (n = 30), MAp44 MO (n = 29), or MAp44 MO coinjected with 250 pg of MAp44 mRNA (n = 19). Lines indicate mean 6 SD. (E) Systolic interval for cMO (n = 27), MAp44 MO (n = 17), and MAp44 MO coinjected with MAp44 mRNA (rescue, n = 21). The lines indicate the mean 6 SD. (F)Asin(E), but displaying the diastolic interval. a, atrium; v, ventricle. ****p # 0.0001 (multiplicity-adjusted), nonparametric one-way ANOVA (Kruskal–Wallis) with the Dunn posttest. n.s., not significant. 3124 COMPLEMENT INHIBITOR MAp44 REGULATES CARDIAC DEVELOPMENT

FIGURE 4. MAp44 knockdown af- fects the migration of cardiac neural crest cells. (A) Whole-mount ISH, using a cardiac neural crest antisense probe (crip2), of 27 hpf old cMO- injected embryos (n =29)(a and c)or MAp44 MO–injected embryos (n =30) (b and d). The arrows in (b)and(d) point to the neural crest cells in the heart of MAp44 MO–injected embryos. (B) Quantification of crip2 staining, as shown in (A), using Fiji. ****p # 0.0001, Mann–Whitney U test. these transcripts. We showed in this article that MAp44-specific CL-LK. Therefore, MASP-3 deficiency would cause the absence knockdown, in the verified absence of MASP-3 perturbation, is of such complexes, whereas MAp44 deficiency would cause in- sufficient to attain a similar effect, potentially suggesting that creased levels. Either scenario could cause aberrant downstream MAp44 is the main effector. However, most human 3MC patients signaling, leading to defects in neural crest cell behavior during present with mutations that do not affect MAp44, only MASP-3, development. Although it is not simple to determine cause and Downloaded from and their clinical pictures are grossly similar to those of patients effect for the observed phenotype, we note that the majority of deficient in MAp44 and MASP-3. Thus, the most parsimonious observed defects could be related to perturbation of neural crest explanation is that MAp44 and MASP-3 act in the same pathway, cell function and behavior, and the remainder could be derived and perturbation of either, or both, is sufficient to cause the phe- secondarily to the heart insufficiency following from dysfunc- notype. Although it remains a hypothesis, we substantiated this tional cardiac neural crest cells. Although patent ductus arteriosi interpretation by the demonstration that MAp44 and MASP-3 have been observed in human 3MC patients (6), more severe http://www.jimmunol.org/ compete for PRM interaction. The high level of expression ob- congenital heart defects have not. We speculate that there may be served during development, particularly in the heart, underscores a strong observational selection bias, because fetuses with severe the importance of the developmental pattern and tissue localiza- congenital heart defects would likely not be carried to term. tion of MAp44. MASP-3 is present during this same time window, To the best of our knowledge, our finding of an involvement of albeit at much lower levels, and we may speculate that tight immune-related molecules in cardiac development is novel, but control of MASP-3 enzymatic activity, in part via MAp44 com- other studies recently indicated additional important develop- petition, is essential. However, the PRM/MASP system is a com- mental functions of immune- and complement-related components. plex protein network with numerous additional components. Three Classical pathway components C1q and C3 are essential for correct collectin-like PRMs were reported in fish: MBL, GalBl (an MBL synaptic pruning in the developing brain (39). It is believed that the by guest on September 27, 2021 homolog with specificity for galactose instead of mannose) (37, C1 complex, through recognition of an undefined motif on de- 38), and CL-K1 (4). Based on analogy with the human PRMs veloping synapses, is capable of delivering complement-mediated and the observations of Rooryck et al. (4), it seems reasonable punishment signals. Deposition of the fragment C3b (generated to suppose that the PRM relevant to the interaction with MAp44 from C3 by cleavage by C3 convertases) and subsequent degra- and MASP-3 during development is CL-K1. We propose a unified dation fragments primes weak synapses for elimination by micro- hypothesis for the observed effects of MASP-3 and MAp44 de- glial phagocytosis. In the absence of functional C1q or downstream ficiency, whereby complexes of CL-LK with MASP-3 are in- components, defective synapse elimination occurs, leading to over- volved in neural crest cell migration, and the composition of these wiring of the neural network. Conversely, aberrant activation of complexes is regulated by competitive interactions of MAp44 with this pathway in the aging brain or its inducement by stress can

FIGURE 5. Competition between MAp44 and MASP-3 for PRM binding. (A) rzMASP-3 was kept at a constant concentration and mixed with increasing concentrations of rzMAp44 and then added to wells coated with MBL. The amount of rzMASP-3 bound to MBL was measured with a zebrafish MASP-3– specific Ab. The y-axis represents signal of bound rzMASP-3, and the x-axis shows the concentration of rzMAp44. (B) Test of the ability of zebrafish MASP-3 to bind human CL-LK, including human MAp44 and human MASP-3 as positive controls. Note that the signals cannot be directly compared between the different proteins because different detection Abs were used. Zebrafish MAp44 could not be assayed in this system because of the un- availability of a detection Ab specific for zebrafish MAp44. (C) Competition between zebrafish MASP-3 and zebrafish MAp44 for binding to human CL- LK. rzMASP-3 was kept at a constant concentration, mixed with increasing concentrations of rzMAp44, and added to wells coated with human CL-LK. The amount of rzMASP-3 bound to CL-LK was measured with a zebrafish MASP-3–specific Ab. The y-axis represents the signal of bound zebrafish MASP-3, and the x-axis represents the concentration of zebrafish MAp44 added. All data are mean 6 SD of duplicate measurements for representative experiments, each repeated twice. The Journal of Immunology 3125 cause a state of overpruning, leading to neurodegeneration (40). 6. Atik, T., A. Koparir, G. Bademci, J. Foster, II, U. Altunoglu, G. Y. Mutlu, S. Bowdin, N. Elcioglu, G. A. Tayfun, S. S. Atik, et al. 2015. Novel MASP1 Complement receptors are also of paramount importance for mutations are associated with an expanded phenotype in 3MC1 syndrome. normal brain development (41). C3a was implicated in neural Orphanet J. Rare Dis. 10: 128–139. stem cell migration and regeneration, because neuronal stem 7. Degn, S. E., J. C. Jensenius, and S. Thiel. 2011. Disease-causing mutations in genes of the complement system. Am. J. Hum. Genet. 88: 689–705. cells express C3aR, and their stimulation by C3a promotes 8. Yongqing, T., P. G. Wilmann, S. B. Reeve, T. H. Coetzer, A. I. Smith, neurogenesis after injury (42), as well as retinal neural regen- J. C. Whisstock, R. N. Pike, and L. C. Wijeyewickrema. 2013. The x-ray crystal eration (43). Finally, complement receptor 2 is expressed on structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, neural progenitor cells and plays a role in hippocampal neu- Malpuech, and Michels (3MC) syndrome. J. Biol. Chem. 288: 22399–22407. rogenesis (44). 9. Hansen, S., L. Selman, N. Palaniyar, K. Ziegler, J. Brandt, A. Kliem, M. Jonasson, M.-O. Skjoedt, O. Nielsen, K. Hartshorn, et al. 2010. Collectin 11 Our model for the MAp44/CL-LK/MASP-3 axis is analogous to (CL-11, CL-K1) is a MASP-1/3-associated plasma collectin with microbial- the scenario for C1q and downstream classical pathway components binding activity. J. Immunol. 185: 6096–6104. in synaptic pruning and neurodegeneration. Just as the absence of 10. Henriksen, M. L., J. Brandt, J. P. Andrieu, C. Nielsen, P. H. Jensen, U. Holmskov, T. J. D. Jørgensen, Y. Palarasah, N. M. Thielens, and S. Hansen. C1q or C3 causes defective pruning leading to overwiring of neural 2013. Heteromeric complexes of native collectin kidney 1 and collectin liver 1 circuits, and age- and stress-associated increases in complement are found in the circulation with MASPs and activate the complement system. tonicity in the brain cause neurodegeneration due to overpruning, so J. Immunol. 191: 6117–6127. 11. Degn, S. E., A. G. Hansen, R. Steffensen, C. Jacobsen, J. C. Jensenius, and may the balance between MAp44 and MASP-3 determine the signal S. Thiel. 2009. MAp44, a human protein associated with pattern recognition tonicity required for correct neural crest cell behavior. Observations molecules of the complement system and regulating the lectin pathway of regarding the role of the classical pathway in neurodevelopment have complement activation. J. Immunol. 183: 7371–7378. 12. Degn, S. E., L. Jensen, T. Olszowski, J. C. Jensenius, and S. Thiel. 2013. Co- only been made in mouse models, and no neurologic defects were complexes of MASP-1 and MASP-2 associated with the soluble pattern- Downloaded from reported for humans deficient in C1q, C1r/C1s, C4, C2, or C3. This recognition molecules drive lectin pathway activation in a manner inhibitable by MAp44. J. Immunol. 191: 1334–1345. may seem surprising given the relatively high compound frequency 13. Lynch, N. J., S.-U.-H. Khan, C. M. Stover, S. M. Sandrini, D. Marston, of such cases. However, the observed defects are subtle even in the J. S. Presanis, and W. J. Schwaeble. 2005. Composition of the lectin pathway of inbred mouse models, indicating that there may be numerous complement in Gallus gallus: absence of mannan-binding lectin-associated serine protease-1 in birds. J. Immunol. 174: 4998–5006. compensatory mechanisms masking an observable phenotype in 14. Nonaka, M., and A. Kimura. 2006. Genomic view of the evolution of the humans. Conversely, in the case of the much rarer 3MC-associated complement system. Immunogenetics 58: 701–713. http://www.jimmunol.org/ defects, it is unlikely that the function of CL-LK/MASP-3/MAp44 is 15. Nagai, T., J. Mutsuro, M. Kimura, Y. Kato, K. Fujiki, T. Yano, and M. Nakao. 2000. A novel truncated isoform of the mannose-binding lectin-associated serine played out through downstream complement signaling because these protease (MASP) from the common carp (Cyprinus carpio). 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Dyn. 203: 253–310. envisioned to contribute to untoward reactions during, for exam- 23. Gouy, M., S. Guindon, and O. Gascuel. 2010. Seaview version 4: a multiplatform ple, age- and disease-associated degenerative disorders. In light of graphical user interface for sequence alignment and phylogenetic tree building. this, it is interesting to note the reported protective function of Mol. Biol. Evol. 27: 221–224. 24. Finn, R. D., P. Coggill, R. Y. Eberhardt, S. R. Eddy, J. Mistry, A. L. Mitchell, MAp44 during ischemia–reperfusion injury (17). S. C. Potter, M. Punta, M. Qureshi, A. Sangrador-Vegas, et al. 2016. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44(D1): D279–D285. Disclosures 25. Henriksen, M. L., K. L. Madsen, K. Skjoedt, and S. Hansen. 2014. Calcium- The authors have no financial conflicts of interest. sensitive immunoaffinity chromatography: gentle and highly specific retrieval of a scarce plasma antigen, collectin-LK (CL-LK). J. 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