A CD46-like Molecule Functional in Teleost Represents an Ancestral Form of Membrane-Bound Regulators of Complement Activation This information is current as of September 27, 2021. Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki, Ryota Nakamura, Tomonori Somamoto and Miki Nakao J Immunol 2015; 194:262-272; Prepublished online 1 December 2014; doi: 10.4049/jimmunol.1303179 Downloaded from http://www.jimmunol.org/content/194/1/262

Supplementary http://www.jimmunol.org/content/suppl/2014/11/29/jimmunol.130317 http://www.jimmunol.org/ Material 9.DCSupplemental References This article cites 63 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/194/1/262.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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A CD46-like Molecule Functional in Teleost Fish Represents an Ancestral Form of Membrane-Bound Regulators of Complement Activation

Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki, Ryota Nakamura, Tomonori Somamoto, and Miki Nakao

In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little

is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a mem- Downloaded from brane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than http://www.jimmunol.org/ control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host–cell protection is an evolutionarily conserved mechanism in regulation of the complement system. The Journal of Immunology, 2015, 194: 262–272.

omplement system is one of humoral innate immune formation of the cytotoxic membrane-attack complex. These po- factors that play a main role in tagging and killing of tent physiological activities are crucial for effective pathogen microbe invaders, as well as modulation of inflammatory elimination, but may potentially cause damage of host cells in C by guest on September 27, 2021 and adaptive immune responses (1, 2). Three distinct activation case of excessive or misdirected activation, resulting in allergic or cascades with different mechanisms in pathogen sensing trigger autoimmune pathology (1, 3–6). the complement activation, as follows: the classical, lectin, and The activation of C3 is therefore tightly controlled by a redun- alternative pathways. These proteolytic cascades form two types dant set of regulatory factors, such as members of the regulator of of enzyme complexes, or C3 convertases (C3bBb and C4b2a), to complement activation (RCA) protein family. RCA proteins are cleave C3 into biologically active fragments, C3a and C3b. Al- characterized by their modular architecture consisting of the though C3a induces inflammatory anaphylatoxic responses, C3b multiple copies of short consensus repeat (SCR) modules, each of can covalently bind target microbes, playing a role as a tag for which is composed of ∼60-aa residues containing typical distri- opsonization and as an anchor for C5 convertase, which triggers bution of four Cys residues and some other consensus residues (1, 7, 8). In humans, secreted RCA proteins such as factor H and C4- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, binding protein (C4bp) and membrane-bound proteins including Kyushu University, Hakozaki, Fukuoka 812-8581, membrane-cofactor protein (MCP or CD46), decay-accelerating Received for publication November 25, 2013. Accepted for publication November 2, factor (DAF or CD55), and complement receptors type 1 and 2 2014. (CR1 or CD35, and CR2 or CD21) have been identified (8). Their This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grant 22380111 (to M.N.). modes of action are categorized into two, as follows: decay ac- The sequences presented in this article have been submitted to the DNA Data Base in celeration of C3 convertase complex and degradation of C3b/C4b Japan/European Molecular Biology Laboratory/GenBank (http://www.ncbi.nlm.nih. fragments as cofactor of the degradation protease, or factor I (8). gov/genbank) under the following accession numbers: zTecrem, AB723859 and cTecrem, AB723858. CR1/CD35 acts as a regulator in both modes and also as a receptor to mediate phagocytosis (9) and immune adherence (10–12), Address correspondence and reprint requests to Dr. Miki Nakao, Laboratory of Ma- rine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, whereas CR2/CD21 functions as a C3d receptor with no regula- Hakozaki, Fukuoka 812-8581, Japan. E-mail address: [email protected] tory role in the complement activation (8). In addition to the roles The online version of this article contains supplemental material. in innate immunity, some membrane-bound RCA, MCP/CD46 Abbreviations used in this article: C4bp, C4-binding protein; CHO, Chinese hamster and CR2/CD21, have been recognized as mediators of adaptive ovary; cTecrem, carp Tecrem; DAF, decay-accelerating factor; GGVB, glucose gel- atin veronal buffer; GVB, gelatin veronal buffer; MCP, membrane-cofactor protein; immune responses, affecting Th1/Th2 balance, Ag presentation, NTR, netrin domain; RCA, regulator of complement activation; rcTecrem, recombi- and B cell activation in mammals (13–18). nant carp Tecrem; SCR, short consensus repeat; STP, Ser/Thr/Pro-rich; Tecrem, Phylogenetically, genes encoding the RCA proteins are classified teleost complement-regulatory membrane protein; zTecrem, zebrafish Tecrem. into two groups, as follows: group 1 composed of factor H and its Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 related factors, and group 2 containing the others in the human www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303179 The Journal of Immunology 263

RCA members (19). Loci of each group form a compact cluster, Identification of Tecrem protein of zebrafish and, in the human genome, the group 1 cluster and the group 2 loci TBLASTN searches of Zebrafish genome database (http://www.ncbi.nlm. are also located in proximity, forming a RCA gene cluster (20– nih.gov/projects/genome/guide/zebrafish/) were conducted using amino 22). Genomic arrangement of the RCA genes and their clustering acid sequence of the human CR1/CD35, CR2/CD21, factor H, DAF/CD55, are diverse among species analyzed to date, such as mice (23, 24), MCP/CD46, and C4bp (a-chain and b-chain). Intron–exon organization chicken (25, 26), frog (27), and teleost, which is the most common was predicted using Softberry program (http://www.softberry.com/berry. phtml). group of the ray-finned bony fish (28–34). In a functional point of view, the secreted RCAs are considered Preparation of cDNA to control excessive complement activation on target surface, Various organs (intestine, gill, kidney, spleen, hepatopancreas, brain, skin, whereas the membrane-bound RCAs should protect host cells from muscle, and heart) were isolated from the and zebrafish. Total misdirected complement attack. Both are in combination essential RNA was extracted from the organs using ISOGEN reagent (Nippon Gene, to prevent autoimmune damages of bystander host tissues from the Tokyo, Japan), according to the manufacturer’s instructions, and 1 mgRNA anaphylatoxic and cytotoxic attack by the complement (1). was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA) and oligo-dT Phylogenetic and genomic lines of evidence have indicated primer. The first-strand cDNA was used as template for expression analysis. that RCA had emerged in cyclostome, the most ancient class of vertebrate (35), and that bony fish are equipped with both group Cloning and sequencing of Tecrem from zebrafish and carp 1 and group 2 RCAs, because factor H–like secreted RCAs and Gene-specific primers (Table I) for 59- and 39-RACE, designed according membrane-bound RCAs have been found in zebrafish genome (33, to the coding sequence of zebrafish Tecrem (zTecrem) predicted from the

34). As for function of RCA in the lower vertebrates, regulatory database, were used for RACE PCR, using SMART RACE cDNA Am- Downloaded from roles in deposition of C3 and degradation of C3b/C4b have been plification Kit (Clontech, Tokyo, Japan) and first-strand cDNA synthesized from total RNA mixture from the intestine, gill, kidney, gonad, spleen, described only for secreted RCAs in lamprey (35) and barred sand hepatopancreas, brain, skin, muscle, and heart. Nested PCR amplifications bass (28–30). No functional data, in contrast, have been reported were performed using Phusion DNA polymerase (New England Biolab, for membrane-bound RCA protein in any lower vertebrate species. Tokyo, Japan) under the following conditions: for first 39-RACE, 30 cycles The present study has aimed at obtaining functional data for of 98˚C for 10 s, 65˚C for 30 s, and 72˚C for 1.5 min; for nested 39-RACE, 30 cycles of 98˚C for 10 s, 60˚C for 20 s, and 72˚C for 1.5 min; for first 59- complement-regulatory roles of membrane-bound RCA molecule http://www.jimmunol.org/ RACE, 30 cycles of 98˚C for 10 s, 55˚C for 30 s, and 72˚C for 1.5 min; and at both mRNA and protein levels. Molecular cloning of membrane- nested 59-RACE, 30 cycles of 98˚C for 10 s, 60˚C for 20 s, and 72˚C for bound RCA, designated teleost complement-regulatory membrane 1.5 min. The final amplified products were subcloned into pGEM-T vector protein (Tecrem), from zebrafish and carp, tissue distribution of (Promega, Madison, WI). More than five clones were independently se- Tecrem mRNA, recombinant Tecrem expression on Chinese hamster quenced on both strands to correct possible PCR errors, using CEQ8800 DNA Analysis system and Dye terminator cycle sequencing kit (Beckman ovary (CHO) cells, and functional assay using the Tecrem-bearing Coulter, Tokyo, Japan). CHO have gained a line of evidence that Tecrem is expressed RACE PCR of carp Tecrem (cTecrem) was performed similarly but ubiquitously in fish tissues playing a protective role from possible using primers based on a carp expressed sequence tag (accession num- host damage by complement. ber EH649440) homologous to zTecrem, and sequenced essentially as

above. by guest on September 27, 2021 Materials and Methods Southern hybridization Materials Genomic DNA (10 mg), isolated from carp erythrocytes according to Mouse IgG1 mAb (AD1.1.10) conjugated with FITC against 63His tag was a standard procedure, was digested to completion at 37˚C for 16 h with 100 purchased from Abcam. Zebrafish were provided by Y. Yoshiura (National U PstI (New England Biolab, Tokyo, Japan) and electrophoresed on a 1% Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan) agarose gel. The DNA fragments were transferred to a Hybond N+ and kept at 27˚C fed with commercial diets. Common carp (Cyprinus membrane and hybridized with digoxigenin-labeled cDNA probe specific carpio) weighing 0.2–1 kg was purchased from a local fish farm. Carp for cTecrem, detected using chemiluminescent substrate of alkaline serum was isolated, as described previously (36). Ginbuna crucian carp phosphatase, closely, as described previously (38). ( auratus langsdorfii, S3N strain), a close relative species of the common carp, weighing ∼50 g, has been maintained in our laboratory, as Tissue expression analysis described elsewhere (37). CHO cells were a gift of H. Tachibana (De- Expression sites of Tecrem were analyzed by RT-PCR with primers GSP2 partment of Bioscience and Biotechnology, Kyushu University) and were and GSP3 for zebrafish and GSP9 and GSP10 for carp (Table I). Zebrafish maintained in Ham’s F12 medium supplemented with 10% FBS. KF-1 GAPDH and carp 40S ribosomal protein S11 subunit cDNA served as cells (Koi carp fin cell line) were maintained in MEM supplemented a positive control for respective species (Table I). After reverse tran- with 2 mM glutamine, 14 mM HEPES, and 10% FBS at 20˚C in 5% CO2. scription from the total RNA with Moloney murine leukemia virus reverse transcriptase and oligo-dT primer, PCRs were performed under the fol- Preparation of anti-CHO carp IgM lowing conditions: initial denaturation for 2 min at 94˚C and 30 cycles of CHO cells (1.0 3 107) were collected in 10 mM EDTA in PBS, washed with 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 1 min, followed by a final extension at 72˚C for 2 min. The amplified products were run on a 2% PBS three times, and suspended in 1 ml PBS. Then the CHO suspension was m emulsified in 1 ml CFA and i.p. injected into carp six times at weekly agarose gel and stained with 0.5 g/ml ethidium bromide. intervals. Next, the carp was bled to separate antiserum. Anti-CHO carp IgM Cloning and sequencing of zTecrem splicing variants was purified from the antiserum by gel filtration on a Superdex 200 pg column (1.6 3 60 cm) equilibrated with 2.5 mM veronal-buffered isotonic cDNA fragments encoding from SCR3 to 39-UT of zTecrem were amplified saline (pH 7.4) containing 2.5% glucose, and stored at 280˚C until use. from various tissues as above and subcloned into pGEM-T vector. Re- sultant clones containing inserts with four different lengths, ranging from Preparation of anti-carp erythrocyte ginbuna crucian carp ∼770 to ∼900 bp, were sequenced, and the nucleotide sequences were serum compared with zebrafish genomic DNA sequences to identify exons, using blastn search at Ensembl genome server (http://asia.ensembl.org/Multi/ Carp erythrocytes were separated from heparinized blood, suspended in Tools/Blast). PBS at 1.0 3 107 cells/ml, and i.p. injected into ginbuna crucian carp four times at weekly intervals at a dose of 0.5 ml suspension/100 g body weight. Flow cytometry The fish were bled 7 d after the last injection, and the sera separated were heat inactivated at 50˚C for 20 min. Sera from three fish, showing ag- Recombinant carp Tecrem (rcTecrem) transformant and mock transformant glutination titer of .128 against carp erythrocytes, were pooled and stored of CHO cells, carp leukocytes, and KF-1 cells were treated with appropriate at 280˚C until use. first Ab and FITC-labeled anti-mouse IgG, and analyzed with Beckman 264 TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS

Table I. Primers used for this study

Primer Name Sequence Information Regarding Primers GSP1 59-AAAGAGTGTGCCGCGATGGA-39 Cloning GSP2 59-GCGGCACCACCATCCATAGAA-39 Cloning/expression analysis (zebrafish) GSP3 59-TTTGCGGCGTGAAATCAGTC-39 Cloning/expression analysis (zebrafish) GSP4 59-GGTGCGGAAGACGATGATGA-39 Cloning GSP5 59-CCCTGATCTCGGCATGTTCTTGT-39 Cloning GSP6 59-GCTTTTGGGTCAACTGGCTTGTG-39 Cloning GSP7 59-GCCCTCAATCTGAATCCAGCACA-39 Cloning GSP8 59-CAAGATGGGCTGTAAGGTGC-39 Cloning Universal primer 59-CTAATACGACTCACTATAGGGC-39 Cloning Nested universal primer 59-AAGCAGTGGTATCAACGCAGAGT-39 Cloning GAPDH forward 59-AGGCCTCTCACAAACGAGGA-39 Expression analysis GAPDH reverse 59-GCATGACCATCAATGACCAG-39 Expression analysis 40S rRNA forward 59-AGACGGGACTACTTGCATTA-39 Expression analysis 40S rRNA reverse 59-ATTGAACCTCACTGTCTTGC-39 Expression analysis GSP9 59-CCCAAGCCTCCTAAAAGGAC-39 Expression analysis (carp) GSP10 59-CAGTTGCCACCTTGTCAGAA-39 Expression analysis (carp) GSP11 59-GTGAAATGTTCAGCACCTCCAG-39 DIG-probe synthesis and genomic PCR GSP12 59-TGACAATGCATTGTGGTGGTTC-39 DIG-probe synthesis and genomic PCR Downloaded from DIG, digoxigenin.

Coulter Epics XL Flow Cytometer equipped with System II software quence confirmation, introduced to Origami B strain. Recombinant C4-1- (Beckman Coulter). Obtained histogram data files were analyzed with NTR and C4-2-NTR of carp were expressed for 24 h at 15˚C in the EXPO 32 MultiCOMP software (Beckman Coulter). presence of 0.1 mM isopropyl b-D-thiogalactoside and purified from the culture supernatant by affinity chromatography on Ni-NTR–agarose col- Establishment of stable CHO transformants expressing umns, essentially following the manufacturer’s instructions (Supplemental http://www.jimmunol.org/ rcTecrem Fig. 1A). The purified rC4-1-NTR and rC4-2-NTR were emulsified in CFA and s.c. A stable CHO cell line expressing rcTecrem (CHO-rcTecrem) was estab- injected into rabbits, four times at weekly intervals. The rabbits were lished as follows: cDNA encoding the cTecrem cDNA lacking putative boosted and 1 wk later bled for separation of antiserum and purification of signal peptides was amplified from the plasmid containing full-length IgG on a HiTrap protein A column (GE Health Science, Tokyo, Japan). cTecrem cDNA, using a sense primer with a 63His tag-coding adapter Anti-carp C4-1 and anti-carp C4-2 are monospecific and showed no cross- and an antisense primer corresponding to the stop codon. A sequence reactivity (Supplemental Fig. 1B, 1C). encoding the cTecrem signal peptide was also PCR amplified. These cDNA segments were cloned into the sites BamHI and EcoRI of pcDNA C3-deposition assay on CHO cells 3.1 Myc/His A vector (Invitrogen). The recombinant plasmid and corre- 7 CHO and CHO-rcTecrem (1.0 3 10 cells) were harvested in PBS con- by guest on September 27, 2021 sponding empty vector were separately introduced to CHO cells using taining 10 mM EDTA (pH 7.4), resuspended in GGVB2+ or EDTA-GVB Lipofectamin and Plus reagent (Invitrogen), and neomycin-resistant at 2.0 3 106 cells/ml, and incubated at 25˚C for 30 min with 200 ml transformed cells were selected by culture in the presence of 0.4 mg/ml 2+ G418 (Invitrogen). normal carp serum diluted 1/10–1/80 with GGVB or EDTA-GVB. Each cell suspension was divided into two aliquots, which were incubated at m Calcein release assay for complement-mediated cytotoxicity room temperature for 30 min with 100 l anti-carp C3 isotype mAb (5C7, 1 mg/ml; 3H11, 10 mg/ml) (40). The cells were then washed with PBS Calcein release cytotoxicity test was performed, as described elsewhere (pH 7.4) in an Ultrafree-MC centrifugal filter tube (Millipore), incubated 4 (35). Briefly, transfected CHO cells (1.0 3 10 cells) were seeded in each with 120 ml 1/60-diluted anti-mouse IgG (whole molecule) F(ab9)2 frag- well of 96-well plates and cultured overnight at 37˚C in 5% CO2. Cells ment–FITC conjugate (Sigma-Aldrich), and analyzed by flow cytometry, were then washed with 0.1 ml PBS three times and labeled with 10 mM as above. calcein-AM (Molecular Probes, Eugene, OR) by incubating at 37˚C for 30 min. The labeled cells were washed three times with 0.1 ml isotonic Preparation of mAb to cTecrem veronal buffered saline (pH 7.4) containing 2.5% glucose, 0.1% gelatin, BALB/c mice were immunized four times at weekly intervals by i.p. 0.15 mM CaCl2, and 1 mM MgCl2 (glucose gelatin veronal buffer injections of CHO-rcTecrem (1.0 3 107 cells) suspended in PBS, and, 2 d 2+ m m [GGVB] ) and incubated with 25 l70 g/ml anti-CHO carp IgM at after a booster injection with the same dose, spleen cells harvested from m 2+ 25˚C for 30 min. Then 25 l normal carp serum diluted with GGVB was the mice were fused with Sp2/O-Ag14 myeloma cells using PEG4000, added, and the plate was incubated at 25˚C for 60 min. The cytolytic re- following a standard method. Hybridomas grown in GIT medium con- m action was halted by adding 100 l GGVB containing 10 mM EDTA, and taining hypoxanthine/aminopterin/thymidine supplements were screened m the plate was centrifuged at 1200 rpm for 5 min. Supernatant (100 l) was by ELISA of the culture supernatants using microtiter plates coated with transferred to a new plate to measure fluorescent intensity with excitation CHO-rcTecrem, CHO, and KF-1 cell lines. Hybridomas positive to both at 495 nm and emission at 535 nm. CHO cells incubated without normal CHO-rcTecrem and KF-1, but negative to CHO, were chosen and cloned carp serum were used for measuring spontaneous calcein release (cont), by the limit-dilution method. and cells lysed with 1% SDS were used for measuring maximum calcein mAb was purified from the culture supernatant using HiTrap protein G release (max). The experiment was performed three times in quadruplicate. (GE Health Science, Tokyo, Japan), according to the manufacturer’s Cytotoxicity was calculated as follows: instructions. À Á ð%Þ¼ 3 ð Þ Cytotoxicity 100 Fsample Fcont Fmax Fcont Immunoprecipitation where F stands for fluorescent intensity. CHO-rcTecrem cells (2.0 3 107 cells) were lysed by sonication in 3 ml PBS containing 3.3% Triton X-100 and 1 mM PMSF on ice, and the lysate Preparation of recombinant carp C4 isotypes and their specific was cleared by centrifugation. The supernatant was, after absorption with Abs 40 ml protein G-Sepharose, incubated with 16 mg anti-cTecrem mAb 1F12 at 4˚C for 1 h. The immune complexes formed were trapped in 40 ml The netrin domain (NTR)-encoding cDNA segments were amplified from protein G-Sepharose, which were washed with 1% Nonidet P-40 in 10 mM respective full-length cDNA clones of carp C4-1 and C4-2 isotypes (39), Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, and 10 mM NaF (pH 7.4), and using primers with adaptor sequences containing BamHI and SacI sites. boiled in 50 mM Tris-HCl, 2% SDS, 5% 2-ME, and 10% glycerol (pH 6.8) The amplified product was subcloned into pCold-I vector and, after se- for SDS-PAGE analysis. The Journal of Immunology 265

Flow cytometric deposition assays of carp C1q, C3-S, C4-1, Phylogenetic relationship of Tecrem and other RCA family and C4-2 on carp erythrocytes members Carp erythrocyte suspension (2 3 108 cells/ml) was incubated with 1/10- Phylogenetic relationship of Tecrems with other RCA protein family diluted anti-carp erythrocyte ginbuna crucian carp antiserum in RPMI members was assessed by drawing a neighbor-joining tree, as shown 1640 at 25˚C for 30 min and washed with RPMI 1640 three times. The in Fig. 3. In fair agreement with a previous report, cTecrem and sensitized carp erythrocyte suspension was incubated at 25˚C for 30 min with 0.5 mg/ml anti-cTecrem mAb 1F12 in RPMI 1640 or with normal zTecrem form a tight cluster branched from a common ancestor of murine IgG1 as a control, washed twice with RPMI 1640, and then with the group 2 genes of vertebrate RCA, suggesting that Tecrem may normal carp serum (1/10 diluted in RPMI 1640) at 25˚C for 60 min to represent an ancestral form of group 2 RCA molecules, such as activate the classical pathway of carp complement. The serum-treated carp MCP/CD46, CR1/CD35, CR2/CD21, DAF/CD55, and C4bps. erythrocytes were incubated with anti-carp C1q A-chain (41), anti-carp C3, anti-carp C4-1, and anti-carp C4-2 rabbit IgG (10 mg/ml) at 4˚C for Duplication of Tecrem gene in carp 30 min, and then with FITC-labeled anti-rabbit IgG (1/200 dilution), and analyzed by flow cytometry, as above. We have examined a possibility of having multiple Tecrem genes in carp genome, because carp is known as a pseudotetraploid spe- Results cies (42, 43), probably generated by allotetraploidization. Thus, copy Molecular cloning of membrane-type RCA from zebrafish and number of Tecrem gene was analyzed by Southern hybridization and carp PCR analysis of carp genome DNA. Genomic Southern hybridization with a 352-bp digoxigenin-labeled probe covering entire SCR3 and Our database search resulted in identification of two RCA- SCR4 gave three bands with approximate size of 16, 12, and 9 kbp encoding loci essentially identical to those reported in the pre- (Supplemental Fig. 2A). Downloaded from ceding paper. One of them, corresponding to a membrane protein In contrast, a genomic DNA segment corresponding to the third previously named ZRC1, was subjected to cDNA cloning from and fourth SCR was PCR amplified using primers 11 and 12 zebrafish mixed organ mRNA. A full-length cDNA sequence (Table I) and subcloned into pGEM-T vector. Sequencing of 12 (2281 bp) containing an open reading frame encoding 385 aa was clones, randomly selected, resulted in identification of three dis- cloned by using RACE PCR. The predicted primary structure tinct sequences, designated types A, B, and C (Supplemental Fig. specified a type I membrane protein composed of, from the N 2B). The appearance of three hybridization bands and three dif- http://www.jimmunol.org/ terminus, a signal peptide, five SCR modules, a short Ser/Pro-rich ferent Tecrem genomic sequences strongly suggests the presence region, a transmembrane region, and a cytoplasmic domain of at least two copies of Tecrem genes in carp genome. (Fig. 1A), thus designated teleost complement-regulatory mem- brane protein (Tecrem). The translated product of zTecrem is Establishment of anti-cTecrem mAb different from that of previously reported ZRC1 in that zTecrem A hybridoma clone, 1F12, producing IgG1 reactive to CHO-rcTecrem possesses a cytoplasmic domain sequence containing a few and KF-1, but not to CHO-mock, was established (Fig. 4A). Spec- phosphorylation sites, whereas ZRC1 lacks the cytoplasmic do- ificity of 1F12 was assessed by ELISA and immunoprecipitation main (34). of the Ag from CHO-rcTecrem. As shown in Fig. 4B, 1F12 The zTecrem amino acid sequence was used to search its carp trapped a 66-kDa polypeptide that was also recognized by anti- by guest on September 27, 2021 homolog by TBLASTN program, gaining two expressed sequence 63His tag. Difference in the observed molecular mass and theo- tag fragments (EG649440 and CA964873) that share ∼50% amino retical value (37.6 kDa) of rcTecrem (including 63His tag, but acid sequence identity with zTecrem. On the basis of these nu- excluding the signal peptide) suggests that the mature rcTecrem cleotide sequences, primers for 59-, 39-RACE PCR were designed, protein expressed on CHO cell is heavily O-glycosylated at the and a complete cDNA sequence (2341 bp) encoding 362 aa of STP-like region, as reported for mammalian CD46 (44). cTecrem was isolated (Fig. 1B). The primary structure of cTecrem Expression of Tecrem protein on carp blood cells was examined specifies a domain structure homologous to that of zTecrem, ex- by flow cytometry using 1F12 mAb. As shown in Fig. 4C and 4D, cept that cTecrem has four SCR modules, followed by a region all the erythrocytes and leukocytes from peripheral blood and rich in Thr and Pro residues in the extracellular domains. Based on leukocytes from the kidney carry Tecrem on their surfaces with no the overall domain architecture, zTecrem and cTecrem molecules significant population of Tecrem-negative cells. are most similar to MCP/CD46, a type I membrane protein con- taining four SCR modules and a Ser/Thr/Pro-rich (STP) region, Expression sites of Tecrem genes when compared with the human RCA homologs. Tecrem expression in various tissues of zebrafish and carp was investigated by RT-PCR. As a result, Tecrem mRNA was detected Splicing variants of zTecrem from all the tissues examined, suggesting that Tecrem is expressed On the result of RT-PCR, we found doublet bands in several tissues, ubiquitously in both species (Fig. 5). Doublet bands of zTecrem suggesting that they are generated by alternative splicing of were detected in several tissues, probably representing the alter- mRNA. To test the possibility, a cDNA region from SCR3 domain natively spliced variants. to 39-UTR of zTecrem was amplified by RT-PCR with the primer set GSP2 and CSP3 and subcloned into pGEM-T vector, and Inhibition of complement activation by Tecrem on CHO cells randomly selected 24 clones were sequenced. As a result, cDNA Predicted complement-regulatory function of Tecrem was examined inserts of four different lengths (823, 773, 916, and 866 bp) were using a cytolytic reaction by carp complement. Calcein-labeled obtained and designated splicing variants 1, 2, 3, and 4, respec- CHO cells were sensitized with carp Abs and attacked by normal tively. Comparison of these nucleotide sequences with that of carp serum mainly via the Ab-dependent classical pathway. Flow a zebrafish genomic DNA contig, NW_001878409, revealed an cytometry of rcTecrem trasformants using anti-His tag Ab showed additional 93-bp exon encoding 31 aa rich in Ser/Thr/Pro, and the that .70% of cells were rcTecrem positive, whereas there is four types of alternative splicing variants differing in lengths of a substantial variation in the expression level (Fig. 6A). the STP region and cytoplasmic region were identified, as sum- Inhibitory effect of Tecrem on complement-mediated cytolysis marized in Fig. 2; zTecrem and ZRC1 (34) correspond to types 1 was assessed by calcein-release assay using CHO-rcTecrem and and 2, respectively. CHO mock sensitized with anti-CHO carp Ab. As shown in 266 TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS Downloaded from

FIGURE 1. Nucleotide and deduced amino acid sequences of Tecrem from zebrafish (A) and carp (B). Asterisks

indicate stop codons. The region http://www.jimmunol.org/ encoding SCR modules, transmembrane (TM), and cytoplasmic region (CYR) is indicated by arrows. The putative phos- phorylation sites for protein kinase C (PKC), casein kinase 2 (CK2), mitogen- activated kinase kinase (MAP2K), and G-coupled receptor kinase (GRK) are shaded. The polyadenylation signals are shown by italic and bold. by guest on September 27, 2021

Fig. 6B, CHO-rcTecrem showed significantly lower level of cy- major isotype of carp C3, because C3-H1 did not show any sig- tolysis by carp complement than that of mock-control cells, sug- nificant binding to CHO under the conditions that allowed com- gesting that Tecrem negatively regulates complement activation. plement activation (data not shown). In the C3-deposition assay, incubation of normal carp serum led to deposition C3-S isotype on the target cells, and this binding was Tecrem inhibits deposition of autologous C3 and C4 after the substantially lower on Tecrem transformant than on mock control, classical pathway activation indicating that Tecrem downregulates deposition of C3-S isotype Regulation of C3 and C4 deposition by Tecrem on autologous cells (Fig. 6C). This assay system did not work for C3-H1, another was assayed using carp erythrocytes sensitized with antiserum The Journal of Immunology 267 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Generation of alternative splicing products of zTecrem. (A) Exon–intron organization of zTecrem gene. Closed and open rectangles show untranslated region and coding region, respectively. Exon and intron size are indicated by number on exon and between exons. Sequences of additional exons are shown below exon–intron organization. (B) Domain structures of zTecrem splicing variants (Sp var. 1–4) and cTecrem. CYR, cytoplasmic region; SP, signal peptide; TM, transmembrane region. raised in ginbuna crucian carp, a close relative of the common carp. number of SCR modules is common in the member of RCA family As shown in Fig. 7, the ginbuna crucian carp IgM was capable of (45, 46), and a minimum functional unit as a binding site usually triggering the classical pathway of carp complement on carp spans only a single or two SCR modules (47–55). Thus, it seems erythrocytes, as evidenced by binding of C1q A-chain, which was unlikely that the difference in the number of SCR modules would not affected by treatment with anti-cTecrem mAb. Deposition of significantly affect functions of Tecrem between the two species. C3 and the two C4 isotypes, C4-1 and C4-2, was also detected on We found four types of splicing variants of zTecrem that differ in the sensitized carp erythrocytes, and they were increased by the serine/threonine-rich and the cytoplasmic regions. cTecrem, treatment with anti-cTecrem, suggesting that Tecrem plays a role which contained a 49-aa residues-long threonine/proline-rich re- in restriction of C3 and C4 deposition on autologous cells. gion and cytoplasmic region, resembled splicing variant 3 of zTecrem in molecular architecture (Fig. 2B). It is intriguing to note Discussion that human MCP/CD46 gene also produces very similar alterna- Our attempts to discover membrane-bound types of RCA protein tive splicing variants differing in the STP-rich region and the have been conducted on zebrafish independently from the group cytoplasmic region (56), implying tight evolutionary conservation that published recently two RCA molecules (ZRC1 and ZRC2), both of this type of splicing variant. In the phylogenetic tree analysis, of which belong to group 2 of RCA family (34). We have tentatively Tecrem branches from the root of group 2 RCA molecules, which named the membrane-bound RCA as Tecrem and found that diverge to mammalian MCP/CD46, DAF/CD55, CR1/CD35, CR2/ zTecrem and ZRC1 are transcripts from the same gene. We have CD21, and C4bp subunits. In addition, whereas tetrapod verte- also cloned its carp homolog for further phylogenetic and func- brates such as frogs, birds, and mammals possess both MCP/ tional studies and revealed that cTecrem is closely similar to CD46-like type I membrane protein RCAs and GPI-anchored zTecrem, but differs in the domain structure such as number of RCAs, there is no indication for the existence of a GPI-anchored SCR modules (4 in carp, 5 in zebrafish). The diversity in the RCA in teleost. Taken together, it is conceivable that Tecrem rep- 268 TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Phylogenetic tree of RCA proteins. Phylogenetic tree was constructed by neighbor-joining method using amino acid sequence of identified two kinds of Tecrem, factor H–like molecule, and RCA proteins (C4bp a-chain, C4bp b-chain, factor H, DAF/CD55, MCP/CD46, CR1/CD35, CR2/CD21, Crry, and Cremp) of higher vertebrates. Database accession numbers for sequences employed are as follows: human factor H, NP_000177; human factor H– related protein 1, NP_002104; human factor H–related protein 2, NP_005657; human factor H–related protein 3, NP_066303; human factor H–related protein 4, NP_006675; human factor H–related protein 5, NP_110414; rat factor H, NP_569093; mouse factor H, NP_034018; mouse factor H–related protein, AAA37416; cow factor H, NP_001029108; wild boar factor H, NP_999446; chicken factor H, XP_426613; Xenopus tropicalis factor H, NP_989218; barred sandbass sand bass cofactor protein, AAA92556; barred sandbass sand bass cofactor-related protein, CAA67355; rainbow trout factor H like, CAF25505; human coagulation factor XIII b subunit, NP_001985; rat coagulation factor XIII b subunit, NP_001099426; mouse coagulation factor XIII b subunit, BAE28851; cow coagulation factor XIII b subunit, NP_001033618; human C4-binding protein a chain, NP_000706; rat C4-binding protein a chain, NP_036648; mouse C4-binding protein, NP_031602; cow C4-binding protein a chain, NP_776677; guinea pig C4-binding protein a chain, BAB39739; chicken C4-binding protein a chain, NP_989995; human C4-binding protein b chain, NP_000707; rat C4-binding protein b chain, AAH88152; cow C4-binding protein b chain, NP_776678; human DAF, NP_000565; rat DAF, BAA88991; mouse DAF, NP_034146; cow DAF, NP_001025474; wild boar DAF, AAG14413; guinea pig DAF, BAA08398; human MCP, NP_002380; rat MCP, NP_062063; mouse MCP, NP_034908; cow MCP, NP_898903; wild boar MCP, NP_999053; rat crry, NP_062174; human complement receptor 1, NP_000564; human complement receptor 1 like, NP_783641; chicken complement-regulatory membrane protein, NP_001028815; human complement receptor 2, NP_001006659; rat, complement receptor 2,NP_001099459; mouse complement receptor 2, NP_031784: lamprey lamprey complement-regulatory protein, BAC77070. resents an ancestral form of membrane-bound group 2 RCA mol- More interestingly from a functional point of view, number of ecules. Identification of membrane-bound RCA in more primitive potential O-glycosylation sites seen on serine/threonine-rich such as agnathans and invertebrates would draw more regions on zTecrem splicing variants and their potency were dif- convincing conclusion. ferent among splicing variants (Supplemental Fig. 3). The dif- The Journal of Immunology 269

FIGURE 5. Expression of Tecrem transcripts in zebrafish and carp tis- sues. (A) zTecrem expression analyzed by RT-PCR. GAPDH was used as positive control. Lane 1, kidney; lane 2, spleen; lane 3, hepatopancreas; lane 4, heart; lane 5, brain; lane 6, intestine; lane 7, gill; lane 8, muscle; lane 9, skin; lane 10, no template control. (B) cTecrem expression detected by RT-PCR. The 40S rRNA S11 was used as a positive control. Lane 1,

head kidney; lane 2, body kidney; lane 3, spleen; lane 4, hepatopancreas; Downloaded from lane 5, heart; lane 6, brain; lane 7, intestine; lane 8, gill; lane 9, skin; lane 10, PBLs; lane 11, no template control. Five zebrafish and three carp individuals were independently analyzed, and representative data are shown.

activation system. In the latter system, anti-carp erythrocyte Ab http://www.jimmunol.org/ FIGURE 4. Specificity of anti-cTecrem mAb (1F12) and detection of raised in a closely related fish species successfully triggered the cTecrem protein on blood cells. (A) Schematic representation of rcTecrem classical activation cascade of carp complement on the carp cells, protein with a 63His tag. (B) ELISA using anti-cTecrem mAb (1F12). and it was demonstrated that anti-cTecrem mAb allowed more CHO, CHO-rcTecrem, and KF-1 cells were cultured in 96-well plate. After deposition of carp C3 and two C4 isotypes on the autologous cells culture, each cell was fixed and used for ELISA using anti-cTecrem mAb by inhibiting the regulatory function of Tecrem. It is unclear which (1F12). (C) Immunoprecipitation using anti-cTecrem mAb 1F12. CHO- rcTecrem cells were lysed in PBS containing 3.3% of Triton X-100 and 1 C3 isotypes were bound to carp erythrocytes, because the anti-carp mM PMSF. Supernatant of lysate was incubated with anti-cTecrem mAb C3 used in this work was polyclonal Ab reactive to all the C3 1F12. Proteins trapped by protein G-Sepharose were electrophoresed on isotypes (57). Taken together, the present results suggest Tecrem 10% polyacrylamide gel. After electrophoresis, gel was stained by Coo- can restrict C3 deposition on the host cell, thereby preventing host by guest on September 27, 2021 massie brilliant blue R-250 (left panel) or used for Western blotting using cell damage by complement-mediated cytotoxicity. It remains to anti-63His tag mAb (right panel). Molecular masses of detected proteins be analyzed whether the mode of inhibitory action of Tecrem is are shown between left and right panels. Ig H and Ig L denote H and L decay acceleration of C3 convertase or C3b degradation as a co- chains, respectively, of mouse Ig. (D) Flow cytometric detection of cTe- factor for factor I. We have cloned two isotypes of complement crem protein on the surface of blood cells. Leukocytes and erythrocytes factor I from carp (58), and are now making their recombinant from carp peripheral blood were treated with anti-cTecrem mAb (1F12), proteins, which will be useful for functional analysis to examine followed by FITC-labeled second Ab treatment, and subjected to flow cytometry. Gray peaks represent negative control samples without anti- detailed inhibitory mechanism of Tecrem. cTecrem mAb. Teleost fish including carp, zebrafish, and trout possess divergent isotypes of C3 differing in binding spectrum to various targets (40, 57, 59). It is particularly interesting to examine how the activation ference seen on the threonine/proline-rich region may have a of different C3 isotypes is regulated by RCA. Preceding studies on functional effect on the Tecrem molecule, because in mammalian comprehensive search of zebrafish RCA genes have revealed only MCP/CD46, O-glycosylation in the STP region controls its protein ZRC1 as a single gene that encodes membrane-bound RCA pro- expression and complement-regulatory function (44, 55). In this tein, suggesting that a Tecrem has a versatile regulatory ability to regard, it should be noted that these splicing variants contained different C3 isoforms in a single teleost species. In the case of different length and potential O-glycosylation sites of threonine/ carp, a pseudotetraploid species, our data suggest the presence of proline-rich regions and may cause functional divergence of duplicated genes of Tecrem, possibly encoding two distinct Tecrem. Tecrem proteins with different inhibition spectra against the di- In mammals, membrane-bound RCA proteins can be categorized versified C3 isotypes. In these contexts, it is interesting to point into two groups: regulators of complement activation by cofactor out that pig DAF fails to act on human C3b, which shares 76% (MCP/CD46) or decay acceleration (DAF/CD55) and complement amino acid identity with pig C3b, resulting in complement- receptors to recognize C3-tagged Ag (CR1/CD35 and CR2/CD21) mediated hyperacute damage of porcine organ transplant in hu- (1, 2). The wide tissue and cellular distribution of Tecrem shown man serum (60, 61). In a single teleost species, such as trout and in the current study suggest its role as complement regulator rather carp, C3 isoforms are as divergent as seen between human and than complement receptor, of which expression would be limited pig, sharing 75–82% identity. If such diverged C3 isoforms can be to phagocytes and other immune cells. regulated by a single kind of RCA protein, the inhibitory mech- Confirming the above assumptions, inhibitory activity of Tecrem anism would be intriguing to analyze to better understand evolu- on complement activation was observed at the protein level through tionary and functional significance of C3 diversity. the experiments using two different assay models, as follows: one Molecular anatomy to identify functional domains essential for employing rcTecrem and another utilizing autologous complement the complement regulation is also an issue to be addressed. In 270 TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS Downloaded from

FIGURE 7. Effect of anti-cTecrem mAb on the deposition of carp C1q, C3, C4-1, and C4-2 to autologous erythrocytes. Carp erythrocytes sensitized with antiserum raised in ginbuna crucian carp were treated with anti- cTecrem mAb (1F12) or with control IgG and then incubated with normal http://www.jimmunol.org/ carp serum to allow activation of the classical complement pathway. Deposi- tion of C1q, C3, C4-1, and C4-2 on the sensitized and complement-activated carp erythrocytes was analyzed by flow cytometry. Gray-filled peak is a neg- ative control without the first Ab. Black and gray lines show carp erythrocytes treated with anti-cTecrem mAb and with control IgG, respectively.

human C3b and C4b (30). It would be interesting to analyze whether Tecrem can regulate both C3b and C4b, and, if yes, which

modules are responsible for the regulation, to gain evolutionary by guest on September 27, 2021 implication of this protein family through vertebrate lineage. Mammalian MCP/CD46 has also been reported to have immu- nomodulatory functions in adaptive immune response. Interaction of C3b with MCP/CD46 expressed on T cells and macrophages has been shown to modify adaptive immune response by controlling T cell activation and inactivation (16–18, 62, 63). The MCP/CD46- like molecular architecture of Tecrem and the presence of motifs for FIGURE 6. Complement-inhibitory activities of cTecrem assayed using cell signaling, such as putative phosphorylation sites by protein CHO cells expressing rcTecrem. (A) Surface expression of rcTecrem on the kinase C, MAPK kinase, and casein kinase 2, in its cytoplasmic recombinant CHO cells revealed by flow cytometry. CHO-rcTecrem and its domain, lead us to hypothesize that Tecrem may also play an im- mock transfectant (CHO-mock) were treated with anti-63His FITC-labeled portant role in linking innate immunity and adaptive immune re- Ab and analyzed by flow cytometry. Gray and black dotted lines show neg- sponse in teleost, especially in balancing Th1/Th2 responses. We ative controls without anti-63His of CHO-rcTecrem and CHO-mock, re- have cloned orthologs of Tecrem from ginbuna crucian carp, a close spectively. Gray-filled peak indicates CHO-mock with anti-63His, showing a species of carp, in which purified B cells and CD4- and CD8- 3 nonspecific signal of anti-6 His. Solid black line shows CHO-rcTecrem positive T cells can be used for analysis of adaptive immune stained with anti-63His. (B) Inhibition of cytotoxicity by carp complement responses (64). Evolutionary conservation of the modulation of against CHO cells by rcTecrem. CHO-rcTecrem and CHO-mock sensitized with carp anti-CHO were incubated with 1/8-diluted normal carp serum in adaptive immune response by CD46-like molecules will be ex- GGVB2+ at 25˚C for 1 h to allow the classical pathway activation, and their plored using the ginbuna crucian carp model. cytotoxicity was measured by the calcein-release assay. The experiment was performed in quadruplicate, and the results are represented as mean 6 SD (p , Acknowledgments C 0.05, Student t test). ( ) Suppression of C3 deposition by rcTecrem on CHO We thank Prof. Hirofumi Tachibana (Department of Bioscience and Bio- cells. CHO-rcTecrem and CHO-mock were attacked by carp complement, as technology, Kyushu University) for providing CHO cells and Dr. Yasutoshi above, but at noncytolytic concentration (1/80 dilution) at 25˚C for 30 min. The Yoshiura (National Institute of Aquaculture) for supplying zebrafish. cells were treated with 1 mg/ml anti-carp C3-S mAb (5C7) and with FITC- labeledsecondAb,andthenanalyzedbyflowcytometrytomeasuredegree of C3 deposition. Gray-filled peak is a negative control without anti–C3-S. Disclosures Gray and black lines show CHO-rcTecrem and CHO-mock, respectively, in- The authors have no financial conflicts of interest. dicating a significant inhibition of C3 deposition onto CHO-rcTecrem. References barred sand bass, a factor H–like soluble RCA has been reported to 1. Walport, M. J. 2001. Complement: first of two parts. N. Engl. J. Med. 344: 1058– show a cofactor activity in factor I–mediated degradation of both 1066. The Journal of Immunology 271

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