, a Human Protein Associated with Pattern Recognition Molecules of the and Regulating the Pathway of Complement Activation This information is current as of October 1, 2021. Søren E. Degn, Annette G. Hansen, Rudi Steffensen, Christian Jacobsen, Jens C. Jensenius and Steffen Thiel J Immunol 2009; 183:7371-7378; Prepublished online 16 November 2009;

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MAp44, a Human Protein Associated with Pattern Recognition Molecules of the Complement System and Regulating the of Complement Activation1

Søren E. Degn,2* Annette G. Hansen,* Rudi Steffensen,‡ Christian Jacobsen,† Jens C. Jensenius,* and Steffen Thiel*

Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 ␮g/ml in Ca2؉-dependent complexes with the soluble pattern recognition molecules. The affinity ؍ for MBL is in the nanomolar range (KD 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. Downloaded from MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate . The Journal of Immunology, 2009, 183: 7371–7378.

he recognition molecules of the innate immune system to the suggestion that MASP-1 cooperates with MASP-2 in gen- include the soluble pattern recognition molecules erating C3 convertase (16). T (sPRMs)3 with collagen-like regions: mannan-binding In this study, we identify an alternative splice variant that encodes http://www.jimmunol.org/ lectin (MBL) and the three ficolins (H-, L-, and M-ficolin). Upon a novel MBL- and ficolin-binding protein, MAp44, and present the recognition of patterns of ligands, they initiate the complement characterization of this protein, including its functional role in con- cascade through activation of proenzymes, MBL-associated serine trolling complement activation through the lectin pathway. proteases (MASPs) (1). The complement system plays a central role in the innate immune system. Upon activation, it facilitates Materials and Methods direct microbial killing, but also acts as a natural adjuvant, en- Analysis of gene structure hancing and directing the adaptive immune response (2). The gene was analyzed using the programs Human Splicing Finder, version The homologous proteases MASP-1 and MASP-3 are encoded 2.3 (D. Hamroun, F. O. Desmet, and M. Lalande, unpublished observa- by guest on October 1, 2021 by the MASP1 gene (3, 4), whereas MASP-2 and a short alterna- tions); polyadq (17); DNA functional site miner, Poly(A) Signal Miner tive splice product, MAp19, are encoded by the MASP2 gene (5, (18); and PolyApred (F. Ahmed, M. Kumar, and G. Raghava, unpublished 6). The three MASPs and MAp19 form homodimers, which asso- observations). ciate with MBL and ficolins through their N-terminal domains (7– RT-PCR and sequencing 10). Activated MASP-2 cleaves the complement factors C4 and C2 Primers were designed to amplify a 497-bp fragment from MAp44 mRNA to generate C3 convertase (9, 11–13). The functions of MASP-1, (forward primer in exon 8; reverse primer in the 3Ј untranslated region MASP-3, and MAp19 remain unresolved, although MASP-1 has (UTR) of the unique exon 9). PCR was performed on cDNA made from been shown to cleave C2 with significant activity (14, 15), leading cell line and tissue RNA (19). The product arising from PCR on human brain cDNA was purified and sequenced. Quantitative real-time RT-PCR (qRT-PCR) *Departments of Medical Microbiology and Immunology and †Medical Biochemis- try, University of Aarhus, Århus, Denmark; and ‡Regional Centre for Blood Trans- mRNA expression levels were quantified in a FirstChoice Human Total fusion and Clinical Immunology, Aalborg Hospital, Ålborg, Denmark RNA Survey Panel (Applied Biosystems/Ambion) comprising RNA from 20 human tissues, using TaqMan chemistry and the ABI Prism 7000 Se- Received for publication July 23, 2009. Accepted for publication September 23, 2009. quence Detection System. The RNA was reverse transcribed using the The costs of publication of this article were defrayed in part by the payment of page Roche One Step RT-PCR system with oligo(dT) primers. TaqMan gene charges. This article must therefore be hereby marked advertisement in accordance expression assays from Applied Biosystems were used for MASP-1 (cat- with 18 U.S.C. Section 1734 solely to indicate this fact. alog no. Hs01111256_m1), MASP-3 (Hs01111266_m1), and MAp44 1 ␤ This work was supported by grants from the Lundbeck Foundation and the Danish (Hs01112777_m1), using 2-microglobulin mRNA (Hs99999907_m1) for Graduate School of Immunology (to S.E.D.). normalization. The relative levels of MASP-1, MASP-3, and MAp44 2 Address correspondence and reprint requests to Dr. Søren E. Degn, Department of mRNA were compared using the delta-delta cycle threshold method. Medical Microbiology and Immunology, The Bartholin Building, Wilhelm Meyers Alle´ 4, University of Aarhus, DK-8000 Århus C, Denmark. E-mail address: Anti-MAp44 Ab [email protected] The C-terminal 19 aa of MAp44 contain the unique C-terminal 17 aa as 3 Abbreviations used in this paper: sPRM, soluble pattern recognition molecule; CDS, well as an N-terminal cysteine for m-maleimidobenzoyl-N-hydroxysuccin- coding sequence; EST, expressed sequence tag; GPC, gel permeation chromatogra- imide ester coupling to keyhole limpet hemocyanin. Two rabbit antisera, phy; HSA, human serum albumin; MASP, mannan-binding lectin-associated serine R74A and R74B, were obtained after immunization regimes, and their Abs protease; MBL, mannan-binding lectin; MRP, MASP-related protein; NHS, normal human serum; pAb, polyclonal Ab; PRM, pattern recognition molecule; qRT-PCR, were affinity purified on peptide-coupled Sepharose 4B beads. These pro- quantitative real-time RT-PCR; SPR, surface plasmon resonance; TRIFMA, time- cedures were conducted by GenScript. resolved immunofluorometric assay; UTR, untranslated region. The Abs were tested on Western blot strips of purified MBL/MASP complexes (containing 30 ␮g of MBL, resulting in ϳ1 ␮g of MBL per Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 strip) or rMAp44 supernatant (containing 300 ␮l of supernatant, 10 ␮l per www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902388 7372 MAp44, A NOVEL REGULATOR OF THE LECTIN COMPLEMENT PATHWAY strip) run on single-well XT-Criterion 4–12% gradient Bis-Tris polyacryl- Competitive binding to MBL amide gel (Bio-Rad) using XT-MOPS running buffer (Bio-Rad) either re- duced or nonreduced as indicated. Precision All Blue prestained marker Fixed concentrations of rMAp44 and rMBL were incubated with increas- (Bio-Rad) was used for the estimation of molecular sizes. The proteins in ing concentrations of rMASP-3. The mixtures were then incubated in man- the gel were blotted to Hybond-ECL membrane (GE Healthcare) in transfer nan-coated microtiter wells. After incubation and washing, the wells were incubated with biotin-labeled polyclonal Ab (pAb) against MAp44 or mAb buffer (25 mM Tris, 0.192 M glycine, 20% v/v ethanol, and 0.1% w/v SDS 3ϩ (pH 8.3)), the membrane was blocked in 0.1% Tween 20 in TBS, and then against MASP-3 (mAb 38.12-3) and developed with Eu -labeled cut into 2.5-mm-wide strips, which were incubated in the wells of Octaline streptavidin. trays (Pateof) with primary Ab in primary buffer (TBS, 0.05% Tween 20, 1 mM EDTA, 1 mg of human serum albumin (HSA)/ml, and 100 ␮gof Effect of MAp44 on activation of the lectin pathway normal human IgG/ml). The strips were washed, incubated with secondary Dilutions of rMAp44 or rMAp19 were made in 10 mM Tris-HCl, 1 M Ab in secondary buffer (TBS/Tween, no azide, 1 mM EDTA, and 100 ␮g ␮ NaCl, 5 mM CaCl2, 100 g HSA/ml, and 0.05% Triton X-100 (pH 7.4) of human IgG/ml), and washed again before being developed with Super- (binding buffer), and rMBL was added to reach 50 ng of MBL/ml. A Signal West Dura Extended Duration Substrate (Pierce). Images were preparation of rMASP-2 (25) was diluted to 5 ng/ml in binding buffer and taken using a charge-coupled device camera (LAS-3000; Fuji) and ana- added to an equal volume of the mixtures above (reaching a final concen- lyzed with the MultiGauge Image Analysis Software supplied with the tration of 25 ng of rMBL/ml, 2.5 ng of rMASP-2/ml, and varying amounts camera. of MAp44 or MAp19). The mixtures were added to mannan-coated wells The primary Abs used for Western blotting were R74A and R74B rabbit to allow binding of MBL complexes. After wash, human complement C4 anti-MAp44 antisera, preimmune sera, as well as the affinity-purified R74A was added and incubated at 37°C. The wells were washed, and a mixture and R74B Abs, mouse monoclonal anti-MASP-1/MASP-3/MAp44 com- of two biotin-labeled mAbs against human C4 was added, followed by mon determinant (1E2; Hycult Biotech), polyclonal rabbit anti-MASP-3 Eu3ϩ-labeled streptavidin and measurement of bound Eu3ϩ. Results were (R32) (3), and polyclonal rabbit anti-MASP-1 (R64) (3). The secondary expressed relative to a standard curve obtained by applying dilutions of a Abs were HRP-conjugated goat anti-rabbit IgG (DakoCytomation) and

standard serum (26). In separate experiments, the amounts of bound Downloaded from HRP-conjugated rabbit anti-mouse Ig (DakoCytomation). MAp44, MAp19, and MASP-2 were measured, as described above. Recombinant proteins Homologies and phylogenetics rMBL was produced, as described (20). MBL/MASP complexes were pu- rified from human plasma, as described (15). MAp44 cDNA in the vector We searched the eukaryote databases for sequences with homologies to pCMV-SPORT6 was purchased from imaGenes (clone IRAK- human MAp44 and assembled a phylogenetic tree. The 1143-nt-long cod- p961F1682Q), and the insert was sequenced. Plasmids encoding MASP-3 ing sequence (CDS) of the human MAp44 mRNA was compared with and MAp19 have been described (3). rMAp44, rMASP-3, and rMAp19 sequences in the nonredundant nucleotide database at National Center for http://www.jimmunol.org/ were produced by transient expression in 293F cells (Invitrogen) and pu- Biotechnology Information using BLASTN (27), identifying full-length rified by affinity chromatography on rMBL-coupled beads by binding in a similar sequences in Macaca fascicularis (gi:90081135), Mus musculus Ca2ϩ-containing buffer and eluting in a buffer containing EDTA and 1 M (gi:26089441), and Rattus norvegicus (gi:55249661). The amino acid se- NaCl. The purity was verified by silver staining of SDS-PAGE gels, and quence of human MAp44 was also blasted against the nonredundant pro- the concentrations were determined by OD measurement and quantitative tein database at National Center for Biotechnology Information using amino acid analysis. BLASTP with default settings, yielding hits for the translated sequences in the aforementioned animals (M. fascicularis, gi:90081136; M. musculus, Assay of MAp44 gi:148665253; and R. norvegicus, gi:55249662), as well as identifying a similar truncated form in Cyprinus carpio, however lacking the 17-aa A sandwich assay was developed, involving capture with mAb 1E2 (re- MAp44 signature (gi:4996234). Genomic alignments and orthologue pre- acting with the N-terminal domains shared by MASP-1, MASP-3, and dictions for the human MASP1 gene were performed using Ensembl (re- by guest on October 1, 2021 MAp44) and detection of bound MAp44 with biotinylated anti-MAp44 Ab, lease 50) (28), identifying homologous transcripts in Pan troglodytes 3ϩ 3ϩ followed by Eu -labeled streptavidin. The amount of Eu in the wells (ENSPTRT00000029309), Macaca mulatta (ENSMMUT00000018241), was read by time-resolved fluorometry (TRIFMA). Canis familiaris (ENSCAFT00000022006), and Danio rerio (ENS MAp44 associated with MBL and ficolins in serum DART00000099500). We further identified the protein named MASP-re- lated protein (MRP) from C. carpio (29), as a MAp44-like protein, as well MBL- or ficolin-containing complexes were extracted by microtiter well- as an orthologous transcript in Ciona intestinalis (30, 31). based affinity chromatography. Wells were coated with 131-1 (mAb anti- The two CDSs of the MASP1/3 gene sequence from Branchiostoma MBL; Bioporta), 4H5 (mAb anti-H-ficolin; Hycult Biotech), GN5 (mAb belcheri (32) were compared with the available Branchiostoma floridae anti-L-ficolin; Hycult Biotech), or monoclonal nonspecific mouse IgG1 genome (DOE Joint Genome Institute; B. floridae version 1.0) (33), iden- (Sigma-Aldrich), and then incubated with diluted normal human serum tifying two homologous regions. In both cases, the exons encoding CCP1 (NHS) and washed; bound material was eluted with SDS-PAGE sample and CCP2 were closely positioned, leaving no space in the intron for an buffer. The samples were analyzed by Western blotting using rabbit anti- extra MAp44-specific exon. In agreement with this, no sequence homolo- MAp44 Abs. MAp44 in complex with MBL or ficolins was also analyzed gous to MAp44 could be identified, and no Branchiostoma expressed se- by TRIFMA. Wells were coated with 131-1, 4H5, GN5, 1E2, or nonspe- quence tags (ESTs) or ESTs from related species aligned to this small cific mouse IgG1, incubated with diluted NHS, and washed, and then rabbit interexonic region. anti-MAp44 or anti-MASP-3 was added, followed by biotinylated swine Xenopus laevis mRNA sequences for MASP1/3a gene product MASP(1) anti-rabbit Ig, and development with Eu3ϩ-labeled streptavidin. Finally, we (gi:6429054) (34) and MASP3a (gi:26005766), and MASP3b gene product also confirmed capture of complexes by developing in a similar set-up MASP3b (gi:26005768) (32) were obtained from GenBank, and their re- using mouse anti-MASP-2/MAp19 mAb (1.3B7) (21). spective CDSs were compared with the draft of the Xenopus tropicalis genome (DOE Joint Genome Institute; X. tropicalis version 4.1), identify- Gel permeation chromatography (GPC) ing only one gene (scaffold_81:2,412,389-2,470,753), which, because it encodes both MASP-1 and MASP-3, we conclude is the MASP1/3a gene. NHS or rMAp44 was subjected to GPC on a Superose 6 column in either 2ϩ The absence of a hit for the MASP3b gene may not be due to the absence Ca -containing buffer or high salt plus EDTA buffer (22). MAp44 was of this gene in tropicalis, as opposed to laevis, but rather due to the in- quantified in the fractions, as described above. Fractions were also ana- completeness of the draft genome of tropicalis. An intron (intron 8) of lyzed for IgM, MBL, and H-ficolin. MASP1/3a could putatively accommodate a MAp44-specific exon, but in Surface plasmon resonance (SPR) silico gene prediction failed to identify an exon. BLAST alignment of X. laevis ESTs vs the genomic sequence did, however, identify a single EST The SPR experiments were similar to those reported (23, 24). Using a (gi:17417909) covering part of exon 5, exons 6–8, and a sequence in intron BIAcore 3000 instrument (GE Healthcare), binding of either rMASP-3 or 8, which we suspect to be a MAp44-specific exon. The EST sequence was rMAp44 was measured on 10940 resonance units of rMBL immobilized on translated revealing a 151-aa uninterrupted sequence. The sequence was a CM5 sensor chip (30 ␮g of rMBL/ml used for derivatization), at a flow BLASTed against National Center for Biotechnology Information’s non- rate of 5 ␮l/min. Equivalent volumes of rMASP-3 and rMAp44 were in- redundant protein database, revealing that the first 142 aa coded for a jected at concentrations from 1 to 30 nM. Data were analyzed by global consecutive CUB and CCP domain similar to MASP-1/-3 from various fitting to a 1:1 Langmuir binding model for several concentrations simul- species, whereas the terminal 9 aa had no obvious similarities. This fits taneously using the BIAevaluation 4.1 software (GE Healthcare). with the sequence representing CUB2-CCP1 and the unique C terminus of The Journal of Immunology 7373 a Xenopus MAp44 orthologue. The genomic region encompassing the MAp44 exon was examined, revealing splice features analogous to the human gene. The aforementioned X. laevis EST was compared with Na- tional Center for Biotechnology Information’s nonhuman, nonmouse EST database using megablast, further identifying four overlapping ESTs, all from X. tropicalis (gi:59237729, gi:71452476, gi:59217533, gi:59210250). The Gallus gallus MASP3 gene (35) was accessed at National Center for Biotechnology Information, and found to have a sufficiently large chicken intron 7 (because the A chain of chicken MASP-1 is only made up of 9 exons as compared with 10 in mammals, the MAp44-specific exon should possibly be found here in chickens) to accommodate an MAp44-specific exon. This chicken intron 7 contained two ESTs, one of them spanning exons 6–8 (gi:82782786), the other only covering exon 8 (gi:14004006). A MAp44 sequence was constructed by joining exons 1–5 from the chicken MASP1 gene with the shared exons 6–7 and the unique exon predicted by EST alignment of gi:82782786. Analogously to the human and Xenopus splice features, the exon has the (c)ag consensus splice ac- ceptor sequence, and two potential branch sites, preceded and followed by polypyrimidine stretches and with no downstream ag dinucleotides until the acceptor ag. To date, no lizard MASP gene has been described, but when we used the sequences of human MASP-1, MASP-3, and MAp44, and X. laevis MASP-1, MASP-3a, MASP-3b, and the putative MAp44, and G. gallus MASP-3 and the putative MAp44 mRNA sequences to search the Anolis Downloaded from carolinensis genome (Broad Institute AnoCar (1.0)), a putative MASP-1/- 3-encoding gene (scaffold_656:284,678-383,614) was identified with no apparent MAp44-specific exon, but a large intron 8. This intron, intron 8, FIGURE 1. Genomic organization, splice pattern, and protein struc- was BLASTed against the EST database, yielding two ESTs: tures. A, Exon-intron structure of the MASP1 gene encoding MASP-1, gi:190286270, which was found to encode a part of exons 6–8, and what MASP-3, and MAp44. Protein-encoding regions are white boxes; 5Ј and 3Ј was suspected to be an MAp44-specific exon, with 3ЈUTR and partial UTRs are gray. Intron sizes are not to scale. The asterisks indicate potential poly(A) tail; and gi:190285980, which was found to encode a small part of N-linked glycosylation sites. Exons 1–8, 10, and 11 encode the identical A http://www.jimmunol.org/ exon 5, exons 6–8, and part of the suspected MAp44-specific exon. The chain of MASP-1 and MASP-3. Exon 12 and exons 13–18 encode the genomic region surrounding this MAp44-specific exon was found to con- serine protease domains of MASP-3 and MASP-1, respectively. The pre- tain the required splice motifs. Based on the sequence alignment of chicken MASP-3 and the identified ESTs with the genomic sequence, the full A. mRNA is spliced differentially to yield the mRNAs encoding the 380-aa- carolinensis MAp44 mRNA sequence was assembled. residue-long MAp44, encompassing the signal peptide, the domains Bos taurus MAp44 was constructed using Model Maker from B. taurus CUB1-EGF-CUB2-CCP1, and 17 extra residues, and the mRNAs for MASP-3 mRNA (NM_001076968.1) based on the following bovine ESTs MASP-1 and MASP-3 encompassing the signal peptide and 6 domains supporting the presence of a MAp44 transcript: gi:112231658 (exons 5–9), (CUB1-EGF-CUB2-CCP1-CCP2, serine protease domain) as well as the gi:87278267 (exons 4–9), gi:82984867 (exons 3–9), and gi:17893086 (ex- activation peptide region. The unique 17 aa of MAp44 are encoded by exon ons 7–9). The ninth exon in B. taurus was further supported by ESTs: 9 located between two of the shared exons of MASP-1 and MASP-3. B, by guest on October 1, 2021 gi:28151761, gi:28152000, gi:45457641, gi:45470175, and gi:87277042. Intron-exon boundaries governing the alternative splice events of MASP- Based on the identified translated protein sequences and translations of 1/-3 vs MAp44 mRNA. The nucleotides surrounding the splice donor and the identified and reconstructed mRNA transcripts, the MAp44 proteins from human and these 12 organisms were aligned using ClustalX version acceptor sites for each of the three introns are indicated. Sequences con- 2.0.10 (36) with default settings and iteration at each alignment step: hu- forming to the gt/ag rule are shown in bold typeface. Exons are shown in man (Homo sapiens: gi:73623026), chimpanzee (P. troglodytes: uppercase, and introns in lowercase letters. The underlined sequence indi- ENSPTRT00000029309), rhesus macaque (M. fascicularis: gi:90081136), cates the predicted optimal branch site (consensus: ctrayy). long-tailed macaque (M. mulatta: ENSMMUT00000018241), cow (B. tau- rus, assembled as described), dog (C. familiaris: ENSCAFT00000022006), mouse (M. musculus: gi:148665253), rat (R. norvegicus: gi:55249662), chicken (G. gallus, assembled as described), lizard (A. carolinensis, as- Because the clones described above were derived from human sembled as described), African clawed frog (X. laevis, assembled as de- fetal brain, we searched for the transcript using a MAp44-specific scribed), zebrafish (D. rerio: ENSDART00000099500), carp (C. carpio: primer set in PCR on human brain cDNA and cDNA from various gi:4996234), and sea squirt (C. intestinalis: gi:198422634). Based on this alignment, a consensus bootstrapped N-J tree was produced, excluding posi- brain-derived cell lines, as well as HeLa and HEK293 cells. PCR tions with gaps and omitting correction for multiple substitutions. The tree was on human brain cDNA yielded a band of the expected size for rooted in FigTree version 1.2.1 using C. intestinalis as outgroup (Fig. 7). Pres- specific MAp44 amplification (supplemental Fig. S1A).4 Sequenc- ence of the characteristic domain-structure (CUB-EGF-CUB-CCP-tail) in all ing this product confirmed its identity with the expected region of assembled and retrieved sequences was verified using Swiss-Prot. MAp44 mRNA. This product was also seen, albeit weaker, with NT2 cells, and even weaker with A172, NHA, and HeLa cells. All Results of these cells also gave a product with a common MASP1 gene A novel MASP1 gene-derived splice product expression primer set (Fig. S1B).

A putative novel mRNA product of the MASP1 gene was identified Features of the gene, splicing, and the resulting mRNA in National Center for Biotechnology Information’s gene database as AL134380.1 and BC039724.1; the former was a 621-bp mRNA The MAp44 splice product is produced from nine exons: the first fragment (H. Blum, S. Bauersachs, W. Mewes, B. Weil, and S. eight exons are shared with the MASP-1 and MASP-3 splice prod- Wiemann, unpublished observations), and the latter was a 2065-bp ucts and code for the CUB1, EGF, CUB2, and CCP1 domains, mRNA (37). The putative protein product encompasses CUB1- whereas the ninth exon is unique to MAp44. An additional adeno- EGF-CUB2-CCP1 (363 aa) of MASP-1/-3 and additional unique sine nucleotide from exon 8 combined with the first 50 nt of exon 17 aa (KNEIDLESELKSEQVTE) C-terminally. The calculated 9 code for the unique 17 aa of MAp44 (Fig. 1). Exon 9 also con- Ј molecular mass of the polypeptide product was 44 kDa, and we tains an extensive 3 UTR, which houses the poly(A) signal. have named this candidate protein mannan-binding lectin-associ- ated protein of 44 kDa, or MAp44. 4 The online version of this article contains supplemental material. 7374 MAp44, A NOVEL REGULATOR OF THE LECTIN COMPLEMENT PATHWAY

Identification of MAp44 in complex with MBL and ficolins in human serum To study MAp44 at the protein level, we purified MBL/MASP complexes from human plasma, we produced rMAp44 in a human cell line, and we raised polyclonal rabbit anti-MAp44 Ab using a peptide representing the C-terminal 19 aa of MAp44. Antiserum and the affinity-purified pAb generated a single band of the ex- pected size of 44 kDa when tested on blots of purified MBL/MASP complex (Fig. S2A) and rMAp44-containing supernatant (Fig. S2B). The MAp44 band was also seen when developing with mAb 1E2 (recognizing an epitope in the common N terminus of MASP- 1/-3/MAp44) (Fig. S2C). To search for the presence of MAp44 in complexes with MBL or ficolins, we used Ab-coated microwells to affinity purify com- plexes from serum, which were then analyzed by Western blotting. Bands at the position expected for MAp44 were seen in the lanes containing the eluate from wells coated with anti-MBL, anti-H- ficolin, and anti-L-ficolin (Fig. 3A), as well as in the lane with

directly loaded MBL/MASP complexes purified conventionally Downloaded from from serum. In separate experiments, we developed identical blots with mAb anti-MASP-2/MAp19 (mAb 1.3B7) to confirm capture of complexes (Fig. S3) and blots of MBL/MASP complexes with mAb 1.3B7 (Fig. S3), mAb 1E2, pAb anti-MASP-1, and pAb anti- MASP-3 (Fig. S2, A and C), to confirm the positions of MAp19, MASP-2, MASP-1, and MASP-3 relative to MAp44. http://www.jimmunol.org/ In addition, we similarly captured MBL and ficolins from serum and probed in situ with anti-MAp44 or anti-MASP-3 Abs. We observed dose-dependent signals in wells coated with anti-MBL, anti-H-ficolin, and anti-L-ficolin, but not in wells coated with mouse IgG (Fig. 3, B and C). As a positive control, we included wells coated with mAb 1E2. We conclude that MAp44 is associ- ated with MBL, H-, and L-ficolin in human serum. FIGURE 2. Expression of mRNA encoding MAp44, MASP-3, and MASP-1 in human tissues. mRNA levels were determined by qRT-PCR. Quantification of MAp44 in human serum by guest on October 1, 2021 The source of the RNA is given below the bars and the relative mRNA level on the y-axis. The values obtained from RNA were set to 1000 We constructed a solid-phase assay for the quantification of U. A–C, Show MAp44, MASP-3, and MASP-1 mRNA levels, respectively. MAp44. Microtiter wells were coated with mAb 1E2, incubated The experiment was performed three times with similar results, each time with samples, and developed with biotinylated rabbit anti-MAp44. using 2 and 20 ng of template cDNA. The samples were diluted in a buffer containing EDTA and high salt, ensuring the dissociation of sPRM/MASP/MAp complexes. The splice donor site of exon 8 and splice acceptor site of exon The MAp44 content was estimated by comparison with highly 10 of MASP-1 and MASP-3 are highly similar to the consensus purified rMAp44. The mean concentrations in serum and EDTA sequences (maggtragt and yyyyyyyyyyynyag, respectively, where plasma from 74 blood donors were 1.38 ␮g/ml (range 0.34–3.00 m ϭ a/c, r ϭ a/g, and y ϭ c/t; Fig. 1B). The acceptor site of exon ␮g/ml) and 0.80 ␮g/ml (range 0.14–2.04 ␮g/ml), respectively. 9 is less conserved, although presenting the crucial terminal ag. Both The distribution of MAp44 conformed to a normal log distribution. splice events conform to the gt/ag rule (38), but only the intron 9/exon 10 junction presents a canonical polypyrimidine tract. SPR analysis of the interaction between MAp44 and MBL A conventional poly(A) site is absent in MAp44 mRNA. How- Using SPR, we determined the strength of the interaction between ever, PolyApred (F. Ahmed, M. Kumar, and G. Raghava, unpub- MAp44 and MBL, and compared it with that of MASP-3 and lished observations) predicts a putative novel poly(A) signal with MBL. The purity of the rMBL has been reported before, and the the sequence ccagac starting at position 1881. The mRNA was rMAp44 and rMASP-3 preparations were deemed pure by silver shown by sequencing to have a 3Ј-terminal poly(A) sequence start- staining of SDS-PAGE gels (Fig. 4A). MBL was coupled to SPR ing at position 1990. chips at two different densities. A SPR chip, activated and blocked, was used for subtraction of the bulk refractive index background. mRNA levels in human tissues A BSA-coated surface served as an extra background control, The levels of mRNA encoding MAp44, MASP-1, and MASP-3 in which gave no higher signal than the blank surface for both ␤ a tissue library were compared with qRT-PCR using 2-micro- MASP-3 and MAp44. Representative sensorgrams are shown for globulin mRNA levels for normalization. The site of highest rel- MAp44 binding and MASP-3 binding (Fig. 4, B and C), yielding ϫ 5 Ϫ1 Ϫ1 ϫ Ϫ5 Ϫ1 ative expression level of MAp44 was the heart, followed by much KDs of 0.6 nM (ka of 1.34 10 s M , kd of 7.82 10 s , ␹2 ϫ 4 Ϫ1 Ϫ1 ϫ Ϫ5 weaker expression in liver, brain, and cervix (Fig. 2A). Apart from of 4.6) and 0.4 nM (ka of 9.3 10 s M , kd of 3.77 10 the heart, the expression profile of MAp44 is similar to that of sϪ1, ␹2 of 30), respectively. The measurements at the other cou- MASP-3 (Fig. 2B). MASP-1 mRNA, in contrast, is predominantly pling density of MBL were in agreement for both MASP-3 and found in liver tissue, with only low copy numbers in cervix, brain, MAp44. The calculated KDs were similar to the 0.8 nM reported placenta, prostate, and bladder (Fig. 2C). for the binding of MASP-2 to MBL (10). The Journal of Immunology 7375 Downloaded from http://www.jimmunol.org/

FIGURE 4. SPR measurements of the interactions between MAp44 and MBL, and between MASP-3 and MBL. A, Silver staining of a SDS-PAGE gel of the purified rMAp44 and rMASP-3 used. B, Sensorgrams for the

interaction of rMAp44 analyte at concentrations from 1 to 30 nM with a by guest on October 1, 2021 fixed amount of rMBL ligand coated on the chip. C, Sensorgrams for the interaction of rMASP-3 analyte at concentrations from 1 to 30 nM on the same surface as in B.

FIGURE 3. Association of MAp44 with MBL and ficolins in serum. A, MBL and ficolins were captured from human serum in microtiter wells complexes are dissociated under high salt plus EDTA conditions. coated with anti-MBL, anti-L-ficolin, or anti-H-ficolin Ab. Nonspecific These findings compare well with those reported for the MASPs monoclonal IgG1 served as control. Bound MBL or ficolins, together with and MAp19 (21). A similar GPC analysis of purified rMAp44 gave associated proteins, were eluted with SDS-sample buffer, applied to non- a peak corresponding to MAp44 in serum under dissociating reducing SDS-PAGE, and analyzed by Western blotting using rabbit anti- MAp44 Ab. Sample identification for each lane is denoted at the top. Pu- conditions. rified MBL/MASP complexes (positive control) were also tested. The Competition between MAp44 and MASP-3 in binding to MBL black lines indicate excision of irrelevant lanes. The Mr in kDa of each band of the marker is given on the right side. The experiment was repeated We assayed the ability of MAp44 to compete with MASP-3 for twice with similar results. B, MBL or ficolins were captured in microtiter binding to MBL. Complexes with MBL were formed in solution, wells, and specific Abs were used to detect MAp44. The capture Abs are and the mixtures were added to mannan-coated wells to allow given below the x-axis. The signal was detected by time-resolved fluorom- MBL to bind. The wells were washed and developed with either etry and is given on the y-axis as counts per second. The error bars indicate anti-MAp44 or anti-MASP-3 Abs. When MAp44 and MASP-3 the SD of duplicate measurements. C, Similar to B, but in this case devel- were incubated simultaneously with MBL, competition between opment was with anti-MASP-3 Ab. the two in binding to MBL was observed (Fig. 6A). We conclude that MAp44 and MASP-3 bind to the same or overlapping sites on MBL. The size distribution of MAp44 in serum NHS was subjected to GPC in an isotonic, Ca2ϩ-containing buffer, MAp44 competes with MASP-2 for binding to MBL and or in a buffer containing EDTA and a high salt concentration (dis- down-regulates C4 cleavage sociating conditions). MAp44 was found to elute as closely over- MASP-2, the C4-activating component of the sPRM/MASP com- lapping twin peaks at ϳ11 and 12 ml in the Ca2ϩ-containing buffer plexes, harbors MBL binding domains that are not identical with (Fig. 5). Under dissociating conditions, a single, symmetrical peak those of MASP-1, MASP-3, and MAp44, but have a similar con- was seen at 14.5 ml, corresponding to an apparent molecular mass figuration. It seemed possible that MAp44 might compete with of ϳ180 kDa. This profile suggests that MAp44 is found in high MASP-2 for binding to MBL. Because such a role was also sug- molecular weight complexes with MBL and ficolins, and that these gested for MAp19, this protein was included in our examinations. 7376 MAp44, A NOVEL REGULATOR OF THE LECTIN COMPLEMENT PATHWAY

FIGURE 5. GPC analysis of the distribution of MAp44 in human se- rum. Serum (100 ␮l) was passed through a Superose 6 column, and frac- tions were analyzed for MAp44 content by TRIFMA. The serum was frac- tionated in an isotonic Ca2ϩ-containing buffer (Œ) or in a high salt and EDTA-containing buffer (conditions dissociating MBL/MASP complexes) (f). Arrows indicate the elution volumes of IgM (970 kDa), IgG (150

kDa), and HSA (67 kDa). The elution positions of MBL and ficolins in Downloaded from Ca2ϩ-containing buffer are also indicated. The experiment was repeated twice with similar results.

We incubated MBL with MAp44 or MAp19 at various concentra- tions, followed by incubation with MASP-2. The complexes were allowed to bind to a mannan-coated surface, followed by incuba- http://www.jimmunol.org/ tion with C4, and finally detection of deposited C4 fragments. MAp44 inhibited C4 deposition, whereas MAp19 did not (Fig. 6B). These observations may be explained by the high affinity for MBL of MAp44, which is very similar to that of MASP-2, whereas that of MAp19 is more than 10-fold lower (ϳ13 nM) (10). We also measured the amount of bound MASP-2 and bound competitor in the complexes in situ. The amount of bound MASP-2 was decreased when adding MAp44, but not when add- ing MAp19 (Fig. 6C). We conclude that MAp44 competes with by guest on October 1, 2021 MASP-2 for binding to MBL, resulting in inhibition of C4 FIGURE 6. MAp44-mediated inhibition of MASP binding and of com- deposition, and hence, inhibition of downstream complement plement activation. A, Competition between MAp44 and MASP-3 for bind- activation. ing to MBL. Constant concentrations of rMAp44 and rMBL were incu- bated with increasing concentrations of rMASP-3. After incubation, the Phylogenetics MBL-containing complexes were captured in microtiter wells coated with mannan, and bound MAp44 was detected with anti-MAp44 Ab and A database search identified orthologs of MAp44 in mammals bound MASP-3 with anti-MASP-3 Ab. The amounts of bound MAp44 (F) (chimpanzee, macaque, dog, mouse, and rat) as well as in bony fish and MASP-3 () are plotted on the left and right-hand side y-axis, respec- (carp and zebrafish). The carp orthologue has been described in the tively. B, Inhibition of MBL/MASP-2 mediated C4 deposition. A mixture literature at the transcript level as MRP (29). A homologue of of rMBL and rMASP-2 was incubated with rMAp44 (F) or rMAp19 (Œ) MRP has been described in sea squirt (a urochordate) at the at increasing concentrations and subsequently incubated in mannan-coated genomic level (30, 31). This prompted us to conduct further da- wells. The wells were next incubated with purified human C4, followed by tabase studies, as delineated in Materials and Methods. MAp44 detection of deposited C4 fragments by anti-C4 Ab. C, Inhibition of was absent in Branchiostoma and present in Xenopus, chicken, and MASP-2 binding to MBL as a function of preincubation with increasing lizard, as well as cow. Its presence/absence could not be deter- concentrations of competitor. A constant concentration of rMBL and rMASP-2 was incubated with increasing amounts of rMAp44 (F)or mined in shark and lamprey, due to the incompleteness of their rMAp19 (Œ). Following incubation in mannan-coated microtiter wells, the genomes. The results are compiled in Table SI, and the resulting wells were developed with anti-MASP-2 Ab. The error bars indicate the phylogenetic tree is shown in Fig. 7. Although it is quite well SD of duplicate measurements. conserved, the hallmark feature of MAp44, i.e., the C-terminal tail, differs radically between fish and mammals. complement system. C1 inhibitor targets C1r/s of the Discussion and MASP-2 of the MBL and ficolin complexes, providing one The surface-associated pattern recognition receptors and the hu- mechanism of control at the level of the recognition complexes moral pattern recognition molecules (PRMs) are pivotal in the (40). However, concomitant inhibition of both the lectin and the induction of immune responses (39). However, uncontrolled acti- classical pathways could be undesirable. In this study, we present vation leads to excessive inflammation, calling for control mech- a selective mechanism for modulation of the activity of lectin- anisms. It is essential to understand not only how immune pathway PRM complexes through the competitive inhibition of responses are initiated, but also how they are modulated and down- MASP-2 activity by MAp44. The relative levels of MASP-2 and regulated after clearance of the innocuous agent or upon activation MAp44 fine-tune the responsiveness of the lectin pathway of com- on self. A number of proteins are involved in the regulation of the plement, and we may speculate that this mechanism contributes to The Journal of Immunology 7377

this truly indicates the formation of higher oligomeric forms than the expected dimer, or whether MAp44 somehow associates with other proteins in a manner not sensitive to high salt and EDTA. The size estimate may be affected by glycosylations, and the mo- lecular masses estimated by size-exclusion chromatography fur- ther rely crucially on the relative shapes of the protein under study compared with the standard proteins used for calibration. This may add to the quite high apparent molecular mass we find, because dimers of MAp19/MASP-2 have been reported to be rather elon- gated (43), and we presume by analogy this could be the case for MAp44. Notably, this finding was consistent between serum MAp44 and purified rMAp44. MAp44 presents the two CUB domains involved in interaction

of MASPs with MBL and the ficolins. The KD values for the bind- ing of MAp44 and MASP-3 to MBL were similar. Because the two proteins harbor identical MBL binding domains, they most likely FIGURE 7. Phylogram based on MAp44 sequence similarity between bind to the same site on MBL. With an affinity for MBL as high as various vertebrate species and a urochordate. A complete alignment of the that of full-length MASPs, it appeared likely that MAp44 com- full-length peptide sequences of MAp44 from human, chimpanzee, rhesus petes with these molecules for binding to MBL, and thus regulates macaque, long-tailed macaque, cow, dog, mouse, rat, chicken, lizard, Af- Downloaded from rican clawed frog, zebrafish, carp, and sea squirt was created using Clust- the activity of the PRM/MASP complexes. Indeed, our results sup- alX version 2.0.10 with default settings and iteration at each alignment port this supposition because MAp44 competed with the binding step. Based on this alignment, a consensus bootstrapped N-J tree was pro- of MASP-2, causing inhibition of MASP-2-mediated complement duced, excluding positions with gaps and omitting correction for multiple activation. MAp44 and MAp19 are both alternative splice frag- substitutions. The tree was rooted in FigTree version 1.2.1 using C. intes- ments of genes encoding full-length proteases. However, whereas tinalis as outgroup. Bootstrap values of 1000 are given on nodes, and MAp44 contains both of the MBL and ficolin binding domains of percentage similarity values to human MAp44 are given in parentheses its protease counterpart, MAp19 contains only CUB1. This makes http://www.jimmunol.org/ after node labels. MAp44 able to bind as strongly as MASP-1/-3 to the PRMs, whereas MAp19 binds weaker than MASP-2, due to a more than 10-fold higher off-rate. Contrary to previous suggestions (44), we the overall level of immunological priming of an individual, with found that MAp19 could not inhibit lectin pathway complement potentially important implications for the balance between mount- activation. ing efficacious responses to pathogens and avoiding autoimmunity. MAp44 is found in many animals. Nagai and colleagues (29) MAp44 comprises the four N-terminal domains common to have demonstrated previously that carp has a duplicated MASP MASP-1 and MASP-3, terminating in an extra sequence of 17 aa gene, both copies of which generate two mRNA species encoding by guest on October 1, 2021 residues encoded by a separate exon. The primary transcript and what they termed the complete MASP-1-like molecule (later rec- mRNA sequences of MAp44 show some peculiarities, most nota- ognized as MASP-3-like) and a related protein, MRP (which we bly a suboptimal 5Ј-splice/branch signal of the intron 8/exon 9 now define as carp MAp44), by alternative polyadenylation and splice site and the absence of a canonical polyadenylation signal, splicing. Although the carp appears to have lost its MASP-1 serine respectively. This may explain why this alternative splice product protease domain-encoding exons by a secondary event (46), we was not discovered earlier by prediction methods, and intriguingly note that the splice pattern of carp MASP-3-like/MRP is analogous indicates that the human genome continues to harbor yet more to that of the human MASP1 gene. Furthermore, a MAp44-like surprises. gene was found in C. intestinalis (a urochordate) based on a mo- We found that like MASP-3, MAp44 is expressed in the liver lecular architecture similar to that of carp MRP, except that it lacks and also in bladder, brain, cervix, colon, and prostate, and at some- a sequence equivalent to the C terminus of carp MRP (30, 31). what lower levels in some other tissues, whereas MASP-1 expres- Interestingly, this C. intestinalis MAp44, unlike all other known sion is largely confined to the liver. Remarkable, and unique for MAp44s and MAp19s, is not generated by alternative polyadeny- MAp44, was a very high expression in the heart. These observa- lation from a MASP gene, but is instead encoded by a distinct gene tions underscore the importance of the mechanism of alternative that lacks a serine protease domain-encoding region. We envisage splicing in regulating expression in various tissues. Their broad that the MAp44-specific exon entered a MASP1-like gene in a expression may indicate local functions of MASP-3 and MAp44, common ancestor, only to have the MASP1-like exon(s) lost in C. different from the one they perform in the circulation. The tissue intestinalis after its divergence from cephalochordates and distribution we observed for MASP-1 and MASP-3 mRNA con- vertebrates. firms previous reports (35, 41). We have described and characterized a novel, evolutionarily Using MAp44-specific Ab, we identified the protein in MBL/ conserved, regulator of the lectin pathway of complement activa- MASP complexes purified from serum, and we find that MAp44 tion. The tissue distribution, as well as the phylogeny, indicates associates not only with MBL, but also with H- and L-ficolin. that MAp44 may have auxiliary functions outside the complement Measurements of MAp44 in serum from blood donors indicated system. Our results may prove to have implications for the regu- a considerable variation at a mean of ϳ1.4 ␮g/ml, comparable lation of inflammatory reactions. with the mean concentrations of MASP-3 (4 ␮g/ml) (42) and MASP-2 (0.5 ␮g/ml) (22). On GPC of serum, all of the MAp44 was found in high molecular weight complexes, which dissociated Acknowledgments in a high salt buffer with EDTA. However, MAp44 unexpectedly Some cDNAs were provided by Anders Lade Nielsen and Jenny Blech- eluted at ϳ180 kDa, corresponding to a tetramer, when analyzed ingberg Friis (Department of Human Genetics, Aarhus University), who by GPC under dissociating conditions. We do not know whether also provided valuable help and suggestions. 7378 MAp44, A NOVEL REGULATOR OF THE LECTIN COMPLEMENT PATHWAY

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